Suppression of cancer

ABSTRACT

Polypeptides for use in suppressing cancer and cancer disorders and methods of treatment using such polypeptides.

This application is a continuation of U.S. application Ser. No. 14/295,798, filed Jun. 4, 2014, which is a continuation of U.S. application Ser. No. 13/125,407, which is a U.S. national stage filing of International Patent Application No. PCT/GB2009/051559, filed on Nov. 17, 2009, which claims the priority of United Kingdom Application No. 0820970.2, filed Nov. 17, 2008. Each of these applications is incorporated herein in their entireties.

The instant application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 7, 2019, is named 286144SeqListing3.txt and is 333,503 bytes in size.

The present invention relates to the suppression of cancer.

Cancers are made up of cells that divide, invade and survive in an aberrant manner in the body. Cancers develop when alterations to the DNA within cells affect the control of cell division and other processes relevant to cell survival. As cancerous tumours grow, they invade the body tissues surrounding them. This is harmful to the body as it damages surrounding normal tissues and has a significant effect on physiological responses. In addition, cancers may spread to other parts of the body and develop into secondary tumours. Assessment of cancer can be undertaken using a variety of tests including CT scan, MRI imaging, PET scans, combined PET-CT scan and ultrasound tests. Generally, the earlier a cancer is detected and confirmed, the better the overall prognosis for the patient.

A wide range of treatments are available for patients diagnosed with cancer. These include a variety of surgical interventions, radiotherapy, chemotherapy and hormone therapies. These treatments may be used in isolation but most commonly they are used in combination with each other. The use of a combination of therapies is dependent on the type of cancer diagnosed with certain cancers (such as breast cancer) commonly requiring multiple therapeutic interventions. There are a variety of new therapeutic approaches under development utilising biological products including antibodies, immunotherapies and vaccination techniques to treat cancer. Additional treatment approaches including the use of novel therapeutics designed to block angiogenesis are under development. Novel approaches including the use of gene therapy are also being investigated as potential therapeutics. However, despite the significant scientific efforts and huge financial commitment to developing new cancer therapies, the prognosis for many patients remains extremely poor.

Breast cancer is the most common cancer in the UK and every year more than 44,000 women are diagnosed with this type of cancer. Worldwide, more than a million women are diagnosed with breast cancer every year. In the UK, approximately 80% of patients will survive for 5 years or longer.

Lung cancer is the 2nd commonest cancer in the UK and over 37,000 people were diagnosed with lung cancer in the UK in 2003. For patients diagnosed with lung cancer, only approximately 20% will survive for at least 1 year after diagnosis and only approximately 6% will live for 5 years or longer after diagnosis.

Colorectal cancer is the 3rd commonest cancer type overall and it is the 2nd commonest affecting women (after breast cancer). Just over 35,000 people were diagnosed with colorectal cancer in the UK in 2003. Of those diagnosed with bowel and rectal cancer in England and Wales, 46% will survive for at least 5 years after their diagnosis.

Pancreatic cancer is the fourth most common cause of death from cancer in the Western world and over 7000 cases are diagnosed in the UK each year. Despite therapeutic advances only 3-4% of those diagnosed with pancreatic cancer survive for 5 years or longer and successful therapeutic intervention is limited to surgery that is applicable to only 15% of patients.

Renal cancer is the 9th commonest cancer type overall with over 7000 new cases diagnosed and greater than 3500 deaths in the UK each year. Around 208,500 new cases of renal cancer are diagnosed in the world each year, accounting for just under 2% of all cancers. It is commoner in men than in women, with a male to female ratio of 1.5:1. Thirty percent of renal cancer patients show signs of advanced renal cell carcinoma at initial diagnosis, with metastases detected in 15-25% of patients at this time.

Cancer represents a worldwide problem that is predicted to grow significantly. In the year 2000, malignant tumours were responsible for 12 percent of the nearly 56 million deaths reported worldwide from all causes. In some countries, more than a quarter of deaths could be attributed to cancer. In 2000, 5.3 million men and 4.7 million women developed a malignant tumour and altogether 6.2 million died from the disease. It is predicted that cancer rates could further increase by 50% to 15 million new cases in the year 2020, (WHO World Cancer Report (IARC, Ed B. W. Stewart and P. Kleihues, 2003).

For a large number of cancers, autocrine signalling (defined as a mode of hormone action in which a hormone binds to receptors on and affects the function of the cell type that produced it) and/or paracrine signalling (defined as a mode of hormone action in which hormone released from endocrine or endocrine-like cells binds to receptors on nearby cells and affects their function), are believed to play a significant role in development of the disease state. For a given cancer, multiple autocrine signalling loops may be implicated in development and maintenance of the cancer state. In addition to providing a stimulus for cell division, autocrine signalling can enhance cell survival acting to protect cells from mechanisms of apoptosis and necrosis and increase the metastatic potential of the cancer cell.

There is therefore a need in the art for new therapies/therapeutics capable of specifically addressing cancer. This need is addressed by the present invention, which solves one or more of the above-mentioned problems.

In more detail, a first aspect of the present invention provides a polypeptide for use in suppressing cancer, wherein the polypeptide comprises:

(i) a non-cytotoxic protease, which protease is capable of cleaving a SNARE protein in a cancer cell;

(ii) a Targeting Moiety (TM) that is capable of binding to a Binding Site on a cancer cell, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the cancer cell, and wherein said cancer cell expresses said SNARE protein; and

(iii) a translocation domain that is capable of translocating the protease from within an endosome, across the endosomal membrane and into the cytosol of the cancer cell;

and with the proviso that the cancer cell is not a neuroendocrine tumour cell;

and with the proviso that the polypeptide is not a clostridial neurotoxin (holotoxin) molecule.

The polypeptide of the present invention is not a naturally-occurring clostridial neurotoxin molecule (also known as clostridial holotoxin). Clostridial holotoxin is one of the most lethal neurotoxins known to man, and, as such, has significant limitations as a therapeutic molecule. Also, in the context of cancer, clostridial holotoxin is associated with undesirable off-site targeting (ie. targeting of non-cancer cells), for example to the neuromuscular junction.

In use, a polypeptide of the invention binds to a cancer cell. Thereafter, the translocation component effects transport of the protease component into the cytosol of the cancer cell. Finally, once inside, the protease inhibits the exocytic fusion process of the cancer cell by cleaving SNARE protein present in the cytosol of the cancer cell. Thus, by inactivating the exocytic fusion apparatus of the cancer cell, the polypeptide of the invention inhibits secretion (eg. of TNF-alpha, acetylcholine, fibroblastic growth factor, gastrin releasing peptide, interleukin-6, VEGF, and/or autocrine mobility factor) therefrom. Accordingly, where said secretion peptides are, for example, involved in autocrine or paracrine signalling, the polypeptide of the invention reduces the signalling drive that the autocrine/paracrine loop would otherwise provide to the cancer cell. Accordingly, the polypeptides of the present invention are capable of suppressing or treating cancer.

The first aspect also embraces a corresponding method for suppressing cancer, said method comprising administering a therapeutically effective amount of a polypeptide of the present invention to a patient. The present invention thereby provides an effective means for treating cancer such as renal, breast, lung, pancreatic, and colorectal (eg. colon).

The polypeptides of the present invention provide a distinct advantage over other therapeutics in that they have the potential to inhibit the secretion of multiple autocrine and paracrine peptides from a targeted cancer cell using a single therapeutic molecule. This reduces significantly the ability of a cancer to adapt to a specific treatment regime by switching to and using alternative autocrine signalling pathways. Thus, the present invention provides a more efficacious therapy by blocking multiple pathways simultaneously.

The principal target cell of the present invention is a cancer cell, for example a cancer cell that secretes one or more hormones (or other bioactive molecules), which, once secreted, bind to a receptor on and affect the function of said cancer cell. Examples of cancer cells targeted by the present invention include: breast, lung, pancreatic, colorectal (eg. colon), adrenal, oesophageal, lymphoma (eg. B-cell; Mantel) cell), leukaemia (eg. multiple myeloma), acute leukaemia, bladder, bone, brain tumours, bowel, cervical, chronic lymphocytic leukaemia, Hodgkin's lymphoma, renal, liver, skin (eg. basal, squamous, melanoma), oropharyngeal, myeloma, prostate (eg. soft tissue sarcoma), gastric, testicular, and uterine.

In one embodiment, the target cell of the present invention is a cancer cell in which the level of SNARE protein expression is undesirable.

For example, undesirable SNARE expression may occur when the expression of a SNARE protein is up-regulated when compared with the same (or very similar) cell type when in a non-cancerous state. In one embodiment, the mRNA encoding a SNARE protein is up-regulated in a target cancer cell. In another (or the same) embodiment, the cellular protein concentration of a SNARE protein is (also) increased in the target cancer cell. When measuring SNARE mRNA or protein levels, a value of ‘one’ may be assigned to the SNARE mRNA or protein concentration in a non-cancerous cell of a normal individual, which cell is otherwise the same (or very similar) to the cell type of the cancer cell in question—this value might be described as a ‘basal expression level’, and is typically an average of expression level values obtained from several cells (of the same or very similar type) and/or from different normal individuals. For example, a lung cell (e.g. a neuroectodermal lung cell, or a lung epithelium cell) in a normal individual may be used for comparison with a small cell lung cancer cell. Thus, up-regulated in the context of the present invention means that the SNARE mRNA or protein level is ‘greater than 1’ when compared with the same cell type in a non-cancerous state in a normal individual. In one embodiment, the SNARE protein or mRNA level is up-regulated by greater than 2- or 3-fold, or by greater than 4- or 5-fold, or by greater than 6- or 7-fold. In another embodiment, the SNARE protein or mRNA level is up-regulated by greater than 8- or 9-fold, or by greater than 10- or 12-fold, or by greater than 15- or 20-fold. In another embodiment, the SNARE protein or mRNA level is up-regulated by greater than 40- or 50-fold, or by greater than 60- or 70-fold, or by greater than 90- or 100-fold.

Alternatively, undesirable SNARE expression may occur when the level of SNARE mRNA and/or protein expression in a cancer cell is substantially normal (ie. substantially the same) when compared with that of a corresponding identical or very similar non-cancerous cell type. In this scenario, however, for example when a patient is suffering from a [prostate, bladder or cervical carcinoma], even a low or normal level of SNARE protein expression may support a secretion from the cancer cells of this type that is paracrine or autocrine in function, and so in the context of the cancer, the SNARE protein is supporting an undesirable secretion. In this scenario, the polypeptides of the present invention are capable of suppressing secretion from said cancer cells, and thereby addressing this need in the art.

In this regard, the present inventors have identified undesirable SNARE expression in a range of cancer cell types. These data are presented as Box Plots in FIGS. 3-47. In summary, the present inventors have confirmed that undesirable SNARE expression such as SNAP-25, syntaxin (eg. syntaxin-1a, syntaxin-2), and VAMP (eg. VAMP-1) is observed in small cell lung cancer cells (eg. when compared with normal lung cells); that undesirable SNARE expression such as SNAP-25, syntaxin (eg. syntaxin-2), VAMP (eg. VAMP-1, VAMP-2, VAMP-3) is observed in lung carcinoid cells; that undesirable SNARE expression such as SNAP-25, syntaxin (eg. syntaxin-2), VAMP (eg. VAMP-1, VAMP-2) is observed in lung carcinoid cells (eg. when compared with normal lung cells); that undesirable SNARE expression such as SNAP-25, syntaxin (eg. syntaxin-2, syntaxin-3), and VAMP (eg. VAMP-1, VAMP-3) is observed in bladder carcinoma cells; that undesirable SNARE expression such as SNAP-25, syntaxin (eg. syntaxin-1a), and VAMP (eg. VAMP-2) is observed in breast cancer cells; that undesirable SNARE expression such as SNAP-25, syntaxin (eg. syntaxin-1a, syntaxin-1b, syntaxin-2, syntaxin-3), and VAMP (eg. VAMP-1, VAMP-2, and VAMP-3) is observed in pancreatic adenocarcinoma cells (eg. when compared with normal pancreatic cells); that undesirable SNARE expression such as SNAP-25, syntaxin (eg. syntaxin-1a, syntaxin-1b, syntaxin-3), and VAMP (eg. VAMP-2, VAMP-3) is observed in prostate carcinoma cells (eg. when compared with benign prostate cells); that undesirable SNARE expression such as SNAP-25, syntaxin (eg. syntaxin-2), and VAMP (eg. VAMP-3) is observed in cervix cancer cells (eg. when compared with normal cervix cells); that undesirable SNARE expression such as syntaxin (eg. syntaxin 3) and VAMP (eg. VAMP-1, VAMP-2, VAMP-3) is observed in B-cell leukaemia cells (eg. when compared with normal B-cell lymphocytes); that undesirable SNARE expression such as syntaxin (eg. syntaxin-1a, syntaxin-3) and VAMP (eg. VAMP-1) is observed (eg. up-regulated) in ovarian cancer cells (eg. when compared with normal ovary cells); that undesirable SNARE expression such as SNAP-25 and VAMP (eg. VAMP-1, VAMP-2, VAMP-3) is observed in gastrointestinal (GI) carcinoid cells (eg. when compared with normal GI cells); and that undesirable SNARE expression such as SNAP-25, syntaxin (eg. syntaxin-1a, syntaxin-2) and VAMP (eg. VAMP-3) is observed (eg. up-regulated) in head and neck cancer cells (eg. when compared with normal head and neck cells). In addition, the present inventors have identified that undesirable SNARE expression such as SNAP-25 and VAMP (eg. VAMP-2) is observed in colon cancer cells; that undesirable SNARE expression such as SNAP-25 is observed in adrenal cancer cells; that undesirable SNARE expression such as syntaxin (eg. syntaxin-2) is observed in skin (eg. melanoma) cancer cells; that undesirable SNARE expression such as syntaxin (eg. syntaxin-3) is observed in leukemia (eg. multiple melanoma) cancer cells; that undesirable SNARE expression such as syntaxin (eg. syntaxin-1a) and VAMP (eg. VAMP-1) is observed in lung adenocarcinoma cells; that undesirable SNARE expression such as VAMP (eg. VAMP-1) is observed in liver cancer cells; that undesirable SNARE expression such as VAMP (eg. VAMP-2) is observed in oesophagus cancer cells; and that undesirable SNARE expression such as VAMP (eg. VAMP-3) is observed in lymphoma cancer cells (eg. B-cell lymphoma and Mantel) cell).

The ‘bioactive’ component of the polypeptides of the present invention is provided by a non-cytotoxic protease. This distinct group of proteases act by proteolytically-cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin)—see Gerald K (2002) “Cell and Molecular Biology” (4th edition) John Wiley & Sons, Inc. The acronym SNARE derives from the term Soluble NSF Attachment Receptor, where NSF means N-ethylmaleimide-Sensitive Factor. SNARE proteins are integral to intracellular vesicle formation, and thus to secretion of molecules via vesicle transport from a cell. Accordingly, once delivered to a desired target cell, the non-cytotoxic protease is capable of inhibiting cellular secretion from the target cell.

Non-cytotoxic proteases are a discrete class of molecules that do not kill cells; instead, they act by inhibiting cellular processes other than protein synthesis. Non-cytotoxic proteases are produced as part of a larger toxin molecule by a variety of plants, and by a variety of microorganisms such as Clostridium sp. and Neisseria sp.

Clostridial neurotoxins represent a major group of non-cytotoxic toxin molecules, and comprise two polypeptide chains joined together by a disulphide bond. The two chains are termed the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa. It is the L-chain, which possesses a protease function and exhibits a high substrate specificity for vesicle and/or plasma membrane associated (SNARE) proteins involved in the exocytic process (eg. synaptobrevin, syntaxin or SNAP-25). These substrates are important components of the neurosecretory machinery.

Neisseria sp., most importantly from the species N. gonorrhoeae, and Streptococcus sp., most importantly from the species S. pneumoniae, produce functionally similar non-cytotoxic toxin molecules. An example of such a non-cytotoxic protease is IgA protease (see WO99/58571, which is hereby incorporated in its entirety by reference thereto). Thus, the non-cytotoxic protease of the present invention is preferably a clostridial neurotoxin protease or an IgA protease.

Turning now to the Targeting Moiety (TM) component of the present invention, it is this component that binds the polypeptide of the present invention to a cancer cell. The TM is preferably a peptide.

The TM binds to a Binding Site on the cancer cell, thereby providing selectivity of the polypeptide to this species of target cell over other cells. By way of example, in gastric cancer, the TM preferably binds to gastric cancer cells in preference to other cells of the stomach, such as those that are not cancerous and/or to other non-cancerous (or cancerous) cells in the body. In this regard, preferred TM embodiments of the present invention include antibodies (eg. monoclonal antibodies, antibody fragments such as Fab, F(ab)′₂, Fv, ScFv, etc., and antibody domains peptides), as well as binding scaffolds, which bind to the receptors identified below. Accordingly, the polypeptides of present invention may include commercially available antibodies or binding scaffolds, which have been designed to achieve specific binding to the target cell or receptor in question. Alternatively, preferred TMs include peptide ligands, such as cytokines, growth factors, neuropeptides, and lectins.

A TM of the present invention binds to a receptor on a cancer cell. By way of example, a TM of the present invention binds to a receptor on a cancer cell selected from the group comprising: a growth hormone-releasing hormone (GHRH, aka GHRF/GRF) receptor; an insulin-like growth factor receptor (e.g. an IGF-1 receptor); a corticotropin releasing factor receptor (e.g. CRHR-2); a gastrin-releasing peptide (GRP) receptor; a bombesin peptide (BB) receptor such as BRS-1, BRS-2, or BRS-3; a growth hormone (GH) receptor; an interleukin receptor (e.g. IL8RA or IL13RA1); a vascular endothelial growth factor (VEGF) receptor; an acetylcholine (ACH) receptor; a somatostatin (SST) receptor, such as SST₁, SST₂, SST₃, SST₄ or SST₅; a cortistatin (CST) receptor; a chemokine (C-X-C motif) receptor such as CXCR4; a neuropilin receptor; a gonadotropin-releasing hormone (GnRH) receptor; a VIP-glucagon-GRF-secretin superfamily receptor, such as a PAC (eg. PAC₁) receptor or a vasoactive intestinal peptide VPAC receptor (e.g. VPAC-1 or VPAC-2) receptor; or an ErbB family member receptor such as an EGF receptor. All of these receptors are over-expressed in cancer cells.

In one embodiment of the present invention, the TM binds to a receptor on a cancer cell selected from the group comprising: FLT such as FLT1, FLT4, FLT3, BRS such as BRS3; CHRN such as CHRNA1, CHRND or CHRNE; EPHA such as EPHA7, EPHA4, EPHA5, EPHA3, EPHA1, EPHA2; EFN such as EFNB3, EFNA1, EFNB2, EFNA3, EFNB1; ErbB such as ERBB2, ERBB4; DLK1, DLL3, FGF such as FGFR1, FGFR3, FGFR2; GRPR; GNRHR; GRPR; GnRHR; JAG such as JAG1 (CD339) or JAG2, IFGR such as IGF1R; leukaemia inhibitory factor receptor (LIFR); NMBR; NOTCHR such as NOTCH3 or NOTCH4; VIPR such as VIPR1; VEGFR such as VEGFR2; SSTR such as SSTR1; NMBR; or PDGFR such as PDGFRA or PDGFRB. All of these receptors are over-expressed in cancer cells.

In one embodiment, the TM is selected from: an adrenomedullin (ADM) peptide, an AM peptide, an autocrine motility factor (AMF) peptide, an amphiregulin peptide, an APRIL (a proliferation-inducing ligand) peptide, an artemin peptide, a betacellulin peptide, a bombesin peptide, a calcitonin receptor (CTR) binding peptide, an ErbB peptide such as an EGF peptide, an endothelin peptide, an erythropoietin peptide (EPO), a fibroblast growth factor (FGF) peptide such as a FGF-5 peptide, an FGF-18 peptide, a bFGF, a FGF8 or a FGF17 peptide, a follicle-stimulating hormone (FSH) peptide, a gastrin peptide, a gastrin releasing peptide (GRP), a glial cell line-defined neurotrophic factor (GDNF) peptide, a ghrelin (GHRL) peptide, a growth hormone-releasing hormone (GHRH) peptide, a granulocyte colony-stimulating factor (G-CSF) peptide, a growth hormone (GH) peptide, a heparin-binding EGF-like growth factor (HB-EGF) peptide, a hepatocyte growth factor/scatter factor (HGF/SF) peptide, an interleukin (IL) such as an IL-1 alpha peptide, an IL-6 peptide, an IL-8 peptide or an IL-10 peptide, an IGF-1 peptide, a stromal cell-derived factor-1 (SDF-1) peptide, a keratinocyte growth factor (KGF) peptide, a proepithelin/granulin peptide, a leptin (LEP) peptide, a LIF peptide, an alpha-melanotropin peptide, a MGSA/GRO-alpha, beta or gamma peptide, a NRG-1alpha peptide, an oxytocin peptide, an osteopontin (OPN) peptide, a neuregulin-1 peptide, a PACAP peptide, a PDGF peptide such as a PDGF-alpha peptide, a PDGF-beta peptide, a PDGF-C peptide or a PDGF-D peptide, a prolactin peptide, a SCF peptide, a somatostatin peptide, a tumour necrosis factor (TNF) peptide such as a TNF-alpha peptide, a TGF-beta peptide, a TGF-beta1 peptide, a VEGF peptide such as a VEGF-C peptide or a VEGF-D peptide, a vasopressin peptide, a VIP peptide, an angiopoietin peptide such as an angiopoietin-1 peptide or angiopoietin-2 peptide, a B-CLL peptide, a BCGF-12KD peptide, a BAFF peptide, a galanin peptide, a GDNF peptide, a GnRH peptide, an IGF-II peptide, a LH peptide, a neurotrophin peptide, a substance P peptide, or a TGF-alpha peptide; as well as truncations and peptide analogues thereof.

In one embodiment, a TM of the present invention binds to a leptin receptor. Suitable examples of such TMs include: leptin peptides such as a full-length leptin peptide (eg. leptin₁₆₇), and truncations or peptide analogues thereof such as leptin₂₂₋₁₆₇, leptin₇₀₋₉₅, and leptin₁₁₆₋₁₂₂.

In another embodiment, a TM of the present invention binds to a ghrelin receptor. Examples of suitable TMs in this regard include: ghrelin peptides such as full-length ghrelin (eg. ghrelin₁₁₇) and truncations or peptide analogues thereof such as ghrelin₂₄₋₁₁₇, and ghrelin₅₂₋₁₁₇; [Trp3, Arg5]-ghrelin (1-5), des-Gln-Ghrelin, cortistatin-8, His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂, growth hormone releasing peptide (e.g. GHRP-6), or hexarelin.

In one embodiment, a TM of the present invention binds to a somatostatin (SST) receptor. By way of example, suitable TMs include: SST peptides and cortistatin (CST)-peptides, as well as peptide analogues thereof such as D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH₂ (BIM 23052), D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-D-Nal-NH₂ (BIM 23056) or c[Cys-Phe-Phe-D-Trp-Lys-Thr-Phe-Cys]-NH₂ (BIM-23268). Further examples include the SST peptides SST-14 and SST-28; as well as peptide and peptide analogues such as: octreotide, lanreotide, BIM23027, vapreotide, seglitide, and SOM230. These TMs are preferred TMs for binding to SST receptors, in particular to sst₁, sst₂, sst₃, sst_(a) and sst₅ receptors.

In another embodiment, a TM of the present invention binds to a gonadotropin-releasing hormone (GnRH) receptor. GnRH is also known as Luteinizing-Hormone Releasing Hormone (LHRH). Examples of suitable GnRH receptor TMs include: GnRHI peptides, GnRHII peptides and GnRHIII peptides, for example the full-length 92 amino acid GnRH precursor polypeptide and truncations thereof such as the decapeptide: pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly CONH2 (SEQ ID NO: 45).

In one embodiment, a TM of the present invention binds to insulin-like growth factor (IGF) receptor. Suitable examples include, for example IGF-1 peptides and IGF-2 peptides.

In one embodiment, a TM of the present invention binds to a VIP-glucagon-GRF-secretin superfamily receptor, such as to a PAC (eg. PAC₁) or to a VPAC (e.g. VPAC-1 or VPAC-2) receptor. Suitable examples of such TMs include pituitary adenylate cyclase-activating peptides (PACAP), vasoactive intestinal peptides (VIP), as well as truncations and peptide analogues thereof.

In one embodiment the TM is a VIP peptide including VIP-1 and VIP-2 peptides, for example VIP(1-28), or a truncation or peptide analogue thereof. These TMs demonstrate a selective binding to VPAC-1. Alternatively, a TM demonstrating a selective binding to VPAC2 may be employed, such as, for example mROM (see Yu et al., Peptides 27 (6) p 1359-66 (2006), which is hereby incorporated by reference thereto). In another embodiment, the TM may be a PACAP peptide, for example PACAP(1-38) or PACAP(1-27), or a truncation of peptide analogue thereof. These TMs are preferred TMs for binding to PAC (eg. PAC-1) receptors.

In one embodiment, a TM of the present invention binds to an interleukin receptor. Suitable TM examples include: IL-1 peptides (e.g. IL-1a, IL-6, IL-18 peptides) and truncations or peptide analogues thereof, IL-2 peptides (e.g. IL-2, IL-3, IL-12, IL-23 peptides) and truncations or peptide analogues thereof, IL-6 and IL-8 peptides and truncations or peptide analogues thereof, and IL-17 peptides (e.g. II-17A, IL-17C peptides) and truncations or peptide analogues thereof.

In another embodiment, a TM of the present invention binds to an NGF receptor. Examples of suitable TMs include full-length NGF, and truncations or peptide analogues thereof.

In one embodiment, a TM of the present invention binds to a vasoactive epidermal growth factor (VEGF) receptor. Examples of suitable TMs include: VEGF peptide (e.g. VEGF-A, VEGF-B, VEGF-C, VEGF-D or VEGF-E and associated splice variants) and truncations or peptide analogues thereof, and placental growth factor (PIGF) and truncations or peptide analogues thereof.

In another embodiment, a TM of the present invention binds to an ErbB receptor. By way of example, the TM is selected from EGF peptides, transforming growth factor-α (TGF-α) peptides, chimeras of EGF and TGF-α, amphiregulin peptides, betacellulin peptides, epigen peptides, epiregulin peptides, heparin-binding EGF (HB-EGF) peptides, neuregulin (NRG) peptides such as NRG1α, NRG1β, NRG2α, NRG2β, NRG3, NRG4 and neuroregulin splice variants, tomoregulin 1 and 2 peptides, neuroglycan-C peptides, lin-3 peptides, vein peptides, gurken peptides, spitz peptides, or keren peptides; as well as truncations and peptide analogues thereof. There are 4 classes of ErbB receptor (termed ErbB1, erbB2, erbB3 and erbB4), which are also referred to as HER receptors. A number of variants of these receptors exist, which arise from alternate splicing and/or cleavage of the full-length receptor (eg EGFR v1 translation starts at aa543; EGFR v11 deletion of aa521-603; EGFR vIII deletion of aa 6-273; EGFRvIII/Δ12-13 deletion of aa 6-273 and 409-520; EGFR vIV deletion of aa 959-1030; EGFR vV truncation at residue 958; EGFR TDM/2-7 tandem duplication of 6-273; EGFR TDM/18-25 tandem duplication of 664-1030; EGFR-TDM/18-26 tandem duplication of 664-1014). In addition, there are four ErbB4 receptor isoforms called erbB4 JM-a, erbB4 JM-b, erbB4 CYT-1 and erbB4 CYT-1.

Preferred TMs bind to ErbB receptors (e.g., ErbB1, ErbB2, ErbB3, ErbB4) and splice variants thereof, in particular the ErbB1 receptor. ErbB TMs may also include proteins which contain EGF motifs with a splice site between the fourth and fifth cysteines within the six cysteine EGF-module, where this module is placed in close proximity to the transmembrane region of the potential ligand. For example, interphotoreceptor matrix proteoglycan-2 (IMP-2), meprin (MEP)1α, MEP1β, mucin (MUC)3, MUC4, MUC12 and MUC17, as well as proteins with a T-knot scaffold such as potato carboxypeptidase inhibitor, and antibodies to ErbB receptors such as cetuximab, ABX-EGF, trastuzumab, or EMD72000. Further examples include chimeras generated by swapping domains (loop sequences and/or connecting amino acids) of different ErbB ligands, such as a mammalian erbB receptor ligand in which the B-loop sequence has replaced by those present in the insect (Drosophila) ErbB ligands, an ErbB ligand in which the C-loop sequence of EGF has been replaced by that of TGFα(44-50), EGF ligands in which one or more domain has been replaced by corresponding sequences in TGFα to create EGF/TGFα chimeras (e.g. E3T, T3E, E4T, T4E, T3E4T, T6E and E3T4E, and EGF chimeras in which the N-terminal TGFα sequence (WSHFND (SEQ ID NO: 46)) or the neuregulin sequence (SHLVK (SEQ ID NO: 47)) has been used to replace the N-terminal EGF sequence C-terminal of the first cysteine residue (NSDSE (SEQ ID NO: 48)), T1E, and Biregulin. Yet further chimeras include EGF in which a domain has been replaced by an EGF-like domain of another protein, such as a blood coagulation, neural development or cell adhesion protein (e.g. Notch 3, Delta 1, EMR1, F4/80, EMR3 and EMR4 receptors).

In another embodiment, a TM of the present invention binds to a growth-hormone-releasing hormone (GHRH) receptor. GHRH is also known as growth-hormone-releasing factor (GRF or GHRF) or somatocrinin. Suitable TM examples of the present invention include the full-length GHRH (1-44) peptide, and truncations or peptide analogues thereof such as:

GHRH(1-29); GHRH(1-37); hGHRH(1-40)-OH; [MeTyr1,Ala15,22,Nle27]-hGHRH(1-29)-NH2; [MeTyr1,Ala8,9,15,22,28,Nle 27]-hGHRH(1-29)-NH2; cyclo(25-29)[MeTyr1,Ala15,DAsp25,Nle27,Orn29+++]- hGHRH(1-29)-NH2; (D-Tyr1)-GHRH (1-29)-NH2; (D-Ala2)-GHRH (1-29)-NH2; (D-Asp3)-GHRH (1-29)-NH2; (D-Ala4)-GHRH (1-29)-NH2; (D-Thr7)-GHRH (1-29)-NH2; (D-Asn8)-GHRH (1-29)-NH2; (D-Ser9)-GHRH (1-29)-NH2; (D-Tyr10)-GHRH (1-29)-NH2; (Phe4)-GHRH (1-29)-NH2; (pCl-Phe6)-GHRH (1-29)-NH2; (N-Ac-Tyr1)-GHRH (1-29)-NH2; (N-Ac-Tyr1, D-Ala2)-GHRH (1-29)-NH2; (N-Ac-D-Tyr1, D-Ala2)-GHRH (1-29)-NH2; (N-Ac-D-Tyr1, D-Ala 2, D-Asp3)-GHRH (1-29)-NH2; (D-Ala2, NLeu27)-GHRH (1-29)-NH2; (His1, D-Ala2, NLeu27)-GHRH (1-29)-NH2; (N-Ac-His1, D-Ala2, N-Leu27)-GHRH (1-29)-NH2; (His1, D-Ala2, D-Ala 4, Nleu27)-GHRH (1-29)-NH2; (D-Ala2, D-Asp3, D-Asn8, NLeu27)-GHRH (1-29)-NH2; (D-Asp3, D-Asn8, NLeu27)-GHRH (1-29)-NH2; [His1, NLeu27]-hGHRH(1-29)-NH2; [NLeu27]-hGHRH(1-29)-NH2; (SEQ ID NO: 49) H-Tyr-Ala-Asp-Ala-lle-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln- Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH2; (SEQ ID NO: 50) H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Met-Ser-Arg-NH2; (SEQ ID NO: 51) H-Tyr-D-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg- Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu- Gln-Asp-Ile-Met-Ser-Arg-NH2; (SEQ ID NO: 52) H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Ile-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln- Glu-Gln-Gly-Ala-Lys-Val-Arg-Leu-NH2; (SEQ ID NO: 53) H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln- Glu-Gln-Gly-Ala-Lys-Val-Arg-Leu-NH2; (SEQ ID NO: 54) His-Val-Asp-Ala-Ile-Phe-Thr-Gln-Ser-Tyr-Arg-Lys- Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Asn-Arg; (SEQ ID NO: 55) His-Val-Asp-Ala-Ile-Phe-Thr-Gln-Ser-Tyr-Arg-Lys- Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln- Glu-Gln-Gly-Ala.

In a further embodiment, the TM binds to a bombesin receptor (eg. BRS-1, BRS-2, or BRS-3). TMs for use in the present invention that bind to a bombesin receptor include: bombesin—a 14 amino acid peptide originally isolated from the skin of a frog (pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂) (SEQ ID NO: 56); and the two known homologues in mammals, namely neuromedin B, and gastrin releasing peptide (GRP) such as porcine GRP-Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-Ala-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (SEQ ID NO: 57), and human GRP-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (SEQ ID NO: 58). Additional TMs include corresponding bornbesin, neurornedin B and GRP truncations as well as peptide, analogues thereof.

In one embodiment, the cancer cell is a lung cancer cell (e.g. a small cell lung cancer cell, a non-small cell lung cancer cell, or a carcinoid lung cancer cell). The present inventors have confirmed that undesirable SNARE expression is observed in lung cancer cells. In this embodiment, the TM ligand binds to a receptor on the lung cancer cell, such as to a receptor selected from: an erythropoietin (EPO) receptor, a VEGF receptor such as a VEGF-3 receptor, an ErbB receptor such as an EGF receptor, an ErbB2 or 3 receptor, an GRP receptor, an ET(A) receptor, a CCK receptor such as a CCK-A or CCK-B receptor, a FLT receptor such a FLT 3 or FLT4 receptor, a CHRND receptor, an EPHA receptor such as an EPHA7, an EPHA4 or an EPHA5 receptor, an EFNA receptor such as an EFNA3 or EFNB3 receptor, a DLK1 receptor, an FGF receptor such as a FGF1 receptor, or a JAG receptor such as a JAG1 (CD339) or a JAG2 receptor. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: erythropoietin (EPO), VEGF such as VEGF-B, VEGF-C, acetylcholine, ephrin peptides such as ephrin-A1, A2, A3, A4 or A5, pro-neuregulin peptides such as pro-neuregulin-2, neuregulin peptides such as neuregulin or NTAK, EGF, TGF such as TGF-beta, GRP, bombesin like peptide, endothelin, PGF, TNF such as TNF-alpha, IL such as IL-6, IL-8, oxytocin, vasopressin, NRG such as NRG-1alpha, bradykinin, HGF, GHRH, FGF such as bFGF, aFGF, FGF-1, FGF-2, NOTCH peptide ligands, FLT3 cytokine, or gastrin; as well as truncations and peptide analogues thereof.

In another embodiment the cancer cell is a bladder cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in bladder cancer cells. In this embodiment, the TM ligand binds to a receptor on the bladder cancer cell, such as to a receptor selected from: an IGF receptor such as an IGF1 receptor, a G-CSF receptor, a VEGF receptor such as a VEGF-2 receptor, an ErbB receptor such as an EGF receptor, a HGF receptor, or a FGF receptor such as a high/low affinity bFGF receptor.

Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: G-CSF, VEGF, HB-EGF, IGF such as IGF-1 or IGF-2, amphiregulin, betacellulin, hepatocyte growth factor (HGF), ErbB such as EGF, TGF such as TGF-apha, IL such as IL-6, granulin peptide such as granulin-4, or FGF such as bFGF; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is a breast cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in breast cancer cells. In this embodiment, the TM ligand binds to a receptor on the breast cancer cell, such as to a receptor selected from: an IGF receptor such as an IGF1 receptor, a VIP receptor such as VIPR-1, a GRP receptor, a NMB receptor, a CXC receptor such as CXCR4, a TNF receptor such as TNF receptor 1 or 2, a VEGF receptor such as VEGFR-2 or VEGFR-3, a neuropilin receptor such as NRP-1 or NRP-2, an integrin receptor such as integrin receptor alpha9beta1, an OB receptor, an ET receptor such as ET-RA or ET-RB, an erythropoietin receptor (EpoR), a TGF-beta receptor, an AMF receptor, or a prolactin receptor. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: TNF such as TNF-alpha, VEGF such as VEGF-A, VEGF-B, VEGF-C, IL such as IL-6, amphiregulin, leptin, endothelin such as ET-1, ET-2, ET-3, FGF such as FGF-2, GHRH, granulin peptide such as granulin-4, erythropoietin, neuromedin peptides such as GRP neuromedin C, neuromedin B, autocrine motility factor (AMF), prolactin, growth hormone such as hGH, IGF such as IGF-1 or IGF-2, VIP peptides, or PACAP peptides such as PACAP-27 or PACAP-38; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is a pancreatic cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in pancreatic adenocarcinoma cells. In this embodiment, the TM ligand binds to a receptor on the pancreatic cancer cell, such as to a receptor selected from: a VIP receptor such as a VIP1 receptor, a VEGF receptor such as a VEGF-1 or VEGF2 receptor, a SST receptor such as a SST1 receptor, a CHRN receptor such as CHRNG, CHRNE, CHRNA1 or CHRND, an IGF receptor such as IGF1R, a BRS receptor such as BRS3, a GnRH receptor, a GRP receptor, a NMB receptor, a type-1 growth factor receptor, an ErbB receptor such as EGFR, a CCK receptor such as CCKB or CCKC, a PDGF receptor such as PDGF-alpha, an ADM receptor, a keratinoctye growth factor (KGF) receptor such as FGFR2b, a type I FGF receptor, a GnRH receptor, a GFRalpha3/RET receptor, or a GDNF receptor. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: ErbB such as EGF, TGF such as TGF-alpha, gastrin, PDGF, adrenomedullin, VEGF, KGF, FGF such as FGF-5 or FGF (a/b), VIP, SST peptides such as SST-14 or SST-28, acetylcholine, neuromedin peptides such as GRP neuromedin C, neuromedin B, PACAP peptided such as PACAP-27 or PACAP-38, IL such as IL-1a, GnRH, bombesin, artemin, GDNF, or IGF such as IGF-1 or IGF-2; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is a prostate cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in prostate carcinoma cells. In this embodiment, the TM ligand binds to a receptor on the prostate cancer cell, such as to a receptor selected from: a CHRN such as a CHRNG or CHRNE receptor, an IGF receptor such as an IGF1 receptor, a VIP receptor such as a VIP1 receptor, a CT receptor, an ErbB receptor such as an EGFR (MR2), a [p75(NTR)] or TrkA receptor, a CC receptor such as a CCR2, a FGF receptor such as FGFR-1 to -4, a KGF receptor, a FSH receptor, a GHS receptor such as GHS-R 1a, a CXC receptor such as CXCR2, a VPAC or PAC receptor such as VPAC1 or PAC1, a CRL receptor, a c-Met/HGF receptor, or a GH receptor. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: calcitonin (CT), TGF such as TGF-alpha, adrenomedullin, acetylcholine, prolactin, IL such as IL-6 or IL-8, NGF, PDF, MCP such as MCP-1, somatostatin, FGF such as FGF1 (acidic FGF), FGF2 (basic FGF), FGF6 or FGF8, EPO, VEGF, KGF, FSH, ghrelin, FGF17, TNF such as TNF-alpha, VIP, PACAP peptides such as PACAP-27 or PACAP-38, AM, HGF, GH, GM-CSF, or IGF such as IGF-1 or IGF-2; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is a cervix or uterine cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in cervix cancer cells. In this embodiment, the TM ligand binds to a receptor on the cervix or uterine cancer cell, such as to a receptor selected from: EPHA receptor such as EPHA5, EPHA7, EPHA4, EPHA3 receptor, a PDGF receptor such as PDGFRA, an EFNA receptor such as EFNA1, an SST receptor such as SSTR1, ab ErbB receptor such as an EGFR, a FLT receptor such as a FLT-1 receptor, a FLK receptor such as a FLK-1 receptor, a VEGF receptor such as VEGFR-3, an ET receptor such as ET(A)R, an IGF receptor such as IGF-1R, or an LIF receptor such as LIFR. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: ErbB such as EGF, TGF such as TGF-alpha, G-CSF, PDGF peptides such as PDGFA or PDGFC, SST peptides such as SST-14 or SST-28, IGF such as IGF-1 or IGF-2, ephrin peptides such as ephrin-A1, A2, A3, A5 or A5, VEGF such as VEGF-C or VEGF-D, granulin peptide such as granulin-4, endothelin such as ET-1, or IL such as IL-6 or IL-1; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is a leukaemia cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in leukaemia cells. In this embodiment, the TM ligand binds to a receptor on the leukaemia cell, such as to a receptor selected from: an FLT receptor such as an FLT1 or 3 receptor, an FGF receptor such as FGFR1, 2 or 3, an EFN receptor such as an EFNB1 or EFNB3 receptor, a JAG receptor such as a JAG1 (cd339) receptor, a PDGF receptor such as PDGFRA or B, EPHA1, EPHA2, a NOTCH receptor such as a NOTCH3 or 4 receptor, an EPHA receptor such as an EPHA7 receptor, a LIF receptor, an EFN receptor such as an EFNA1 or 3 receptor, a DLL receptor such as a DLL3 receptor, an ErbB receptor such as an ErbB2 or 3 receptor, a KDR receptor, a GHS receptor such as GHS-R type 1a, an IL receptor such as IL-1 receptor type II, or an IGF receptor such as an IGF1R. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: IGF such as IGF-1 or IGF-2, VEGF peptides such as VEGFB, ghrelin, APRIL, pro-neuregulin peptides such as pro-neuregulin-2, neuregulin peptides, NTAK, FGF such as bFGF, aFGF or FGF-1, NOTCH peptides such as NOTCH1, 2 or 3, PGF peptides, PDGF peptides such as PDGFA, PDGFB or PDGFD, ephrin peptides such as ephrin-A (1-5), LIF, GP30 ligand, erythropoietin, JAG peptides such as JAG-1 or JAG-2, Delta-1, IL such as IL1-alpha, or GM-CSF; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is an ovary cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in ovarian cancer cells. In this embodiment, the TM ligand binds to a receptor on the ovary cancer cell, such as to a receptor selected from: an ErbB receptor such as an ErbB2, 3 or 4 receptor, an FGF receptor such as FGFR3, a JAG receptor such as a JAG2 receptor, an EPH receptor such as an EPHA1 or 2 receptor, an IGF receptor such as an IGF1R, a GRP receptor, an EFN receptor such as an EFNB3, A1 or B2 receptor, or a GNRH receptor. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: NRG peptides such as NRG-2 or NRG-3, or heparin-binding EGF-like growth factor peptides; neuregulin peptides, GP30 peptide, NOTCH peptides, granulin peptides such as granulin-4, ephrin-A peptides such as ephrin A1-A5, IGF peptides such as IGF-1 or IGF-2, NTAK, GRP, or GnRH; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is a bone cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in bone cancer cells. In this embodiment, the TM ligand binds to a receptor on the bone cancer cell, such as to a receptor selected from: an IGF receptor such as an IGF-IR, a GHRH receptor, or a FGF receptor such as a bFGF receptor. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: PDGF such as PDGF-alpha or PDGF-beta, TGF such as TGFbeta1, IGF such as IGF-1, GHRH, FGF such as bFGF, G-CSF, or IL such as IL-6; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is a brain cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in brain cancer cells. In this embodiment, the TM ligand binds to a receptor on the brain cancer cell, such as to a receptor selected from: an EPO receptor, receptor 78 kDa glycoprotein (gp78), an ErbB receptor such as EGFR, as PDGF receptor such as PDGFR-alpha, a CRL receptor such as CRLR/RAMP2 or CRLR/RAMP3, a neuropilin receptor such as NRP-1 or NRP-2, a CXC receptor such as CXCR4, a VEGFR such as VEGFR-1, an IGFR such as IGF1R, a GDNF receptor such as GDNFR-alpha 1, or a MET receptor. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: erythropoietin (EPO) peptides, IGF peptides, autocrine motility factor (AMF) peptides, GDNF peptides, HB-EGF peptides, TGF peptides such as TGF-alpha, PDGF peptides such as PDGFA, PDGF-B, PDGF-C or PDGF-D, neuregulin peptides such as neuregulin-1, adrenomedullin, IL peptides such as IL-6, scatter factor/hepatocyte growth factor (SF/HGF) peptides, granulin peptides such as granulin-4, VEGF peptides such as VEGF-A, or TGF peptides such as TGF beta 1 or TGF beta 2; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is a colorectal cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in colorectal cancer cells In this embodiment, the TM ligand binds to a receptor on the colorectal cancer cell, such as to a receptor selected from: an IGF receptor such as IGF-R1, an ErbB receptor such as EGFR, a GRP receptor, an IL receptor such as IL6-R, a CCK receptor such as CCK-B receptor, or a prolactin receptor. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: FGF such as FGF18, IGF such as IGF-1, TGF such as TGF-alpha, GRP, IL such as IL6, ErbB such as EGF, gastrin, or prolactin; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is a liver cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in liver cancer cells In this embodiment, the TM ligand binds to a receptor on the liver cancer cell, such as to a receptor selected from: a TrkA (NGF) receptor, an IGF receptor such as IGF-IR, a HGF receptor such as a HGF-Met receptor, a c-met receptor, a gp78 receptor, or an ErbB receptor such as an EGFR. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: NGF, IL such as IL-6 or IL-8, IGF such as IGF-1 or IGF-2, HGF, SF/HGF, hepatopoietin (HPO), AMF, TGF such as TGF-beta or TGF-alpha, LIF or PDGF; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is a skin cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in skin cancer cells In this embodiment, the TM ligand binds to a receptor on the skin (eg. basal, melanoma, squamous) cancer cell, such as to a receptor selected from: a FGF receptor such as FGFR-1, a c-kit receptor, a VEGF receptor such as VEGFR-2, a c-Met receptor, or a melanocortin receptor such as a melanocortin-1 receptor. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: IL such as IL8 or IL-10, FGF such as bFGF, SCF, VEGF such as VEGF-A, ErbB such as EGF, EPO, osteopontin (OPN), TGF such as TGF-beta, MGSA/GRO such as MGSA/GRO alpha, beta or gamma, HGF/SF, alpha-melanotropin, amphiregulin, or AMF; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is a Kaposi sarcoma cancer cell. The present inventors have confirmed that undesirable SNARE expression is observed in Kaposi sarcoma cancer cells In this embodiment, the TM ligand binds to a receptor on the Kaposi sarcoma cell, such as to a receptor selected from: a PDGF receptor such as PDGFRA, a c-kit receptor, a TGF-beta receptor such as TGFR-1, an endothelin receptor such as ETA-R, a chemokine receptor such as CXCR3 or CCRL2, a VEGF receptor such as VEGFR-2, an IGF receptor such as IGF-IR, or an angiopoietin receptor such as TIE2. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: PDGFa, IGF-1, Ang2, IL such as IL6, VEGF such as VEGF-A, endothelin-1, TGF such as TGF-beta, CXCL11, CCL8/14; as well as truncations and peptide analogues thereof.

In one embodiment the cancer cell is a renal cancer cell. In this embodiment, the TM ligand binds to a receptor on the renal cancer cell, such as to a receptor selected from: an IGF receptor such as IGF-IR, a VEGF receptor, a CXC receptor, or an ErbB receptor such as an EGFR. Preferred TMs in this embodiment include the corresponding natural ligand to said receptors, as well as truncations and peptide analogues thereof. Examples include: IL such as IL-6 or IL-8, IGF such as IGF-1 or IGF-2, TGF such as TGF-beta or TGF-alpha, VEGF such as VEGF-A, chemokines such as CXCL12; as well as truncations and peptide analogues thereof.

The above embodiments describe particular cancers in which the present inventors have demonstrated undesirable SNARE expression (eg. up-regulated. SNARE expression). The present invention is not, however, limited to said specific cancer types, and embraces all cancer types in which SNARE expression occurs.

The above-described undesirable SNARE expression (eg. up-regulation) in cancer cells may optionally be linked to an up-regulation of one or more specific receptor types present on the cancer cells in question. In this regard, the present inventors have identified particular receptor types (detailed above), which are up-regulated in the same cells in which undesirable SNARE expression is observed (eg. up-regulated).

Polypeptide Preparation

The polypeptides of the present invention comprise 3 principal components: a ‘bioactive’ (ie. a non-cytotoxic protease); a TM; and a translocation domain. The general technology associated with the preparation of such fusion proteins is often referred to as re-targeted toxin technology. By way of exemplification, we refer to: WO94/21300; WO96/33273; WO98/07864; WO00/10598; WO01/21213; WO06/059093; WO00/62814; WO00/04926; WO93/15766; WO00/61192; and WO99/58571. All of these publications are herein incorporated by reference thereto.

In more detail, the TM component of the present invention may be fused to either the protease component or the translocation component of the present invention. Said fusion is preferably by way of a covalent bond, for example either a direct covalent bond or via a spacer/linker molecule. The protease component and the translocation component are preferably linked together via a covalent bond, for example either a direct covalent bond or via a spacer/linker molecule. Suitable spacer/linked molecules are well known in the art, and typically comprise an amino acid-based sequence of between 5 and 40, preferably between 10 and 30 amino acid residues in length.

In use, the polypeptides have a di-chain conformation, wherein the protease component and the translocation component are linked together, preferably via a disulphide bond.

The polypeptides of the present invention may be prepared by conventional chemical conjugation techniques, which are well known to a skilled person. By way of example, reference is made to Hermanson, G. T. (1996), Bioconjugate techniques, Academic Press, and to Wong, S. S. (1991), Chemistry of protein conjugation and cross-linking, CRC Press, Nagy et al., PNAS 95 p 1794-99 (1998). Further detailed methodologies for attaching synthetic TMs to a polypeptide of the present invention are provided in, for example, EP0257742. The above-mentioned conjugation publications are herein incorporated by reference thereto.

Alternatively, the polypeptides may be prepared by recombinant preparation of a single polypeptide fusion protein (see, for example, WO98/07864). This technique is based on the in vivo bacterial mechanism by which native clostridial neurotoxin (i.e. holotoxin) is prepared, and results in a fusion protein having the following ‘simplified’ structural arrangement:

NH₂-[protease component]-[translocation component]-[TM]-COOH

According to WO98/07864, the TM is placed towards the C-terminal end of the fusion protein. The fusion protein is then activated by treatment with a protease, which cleaves at a site between the protease component and the translocation component. A di-chain protein is thus produced, comprising the protease component as a single polypeptide chain covalently attached (via a disulphide bridge) to another single polypeptide chain containing the translocation component plus TM.

Alternatively, according to WO06/059093, the TM component of the fusion protein is located towards the middle of the linear fusion protein sequence, between the protease cleavage site and the translocation component. This ensures that the TM is attached to the translocation domain (ie. as occurs with native clostridial holotoxin), though in this case the two components are reversed in order vis-à-vis native holotoxin. Subsequent cleavage at the protease cleavage site exposes the N-terminal portion of the TM, and provides the di-chain polypeptide fusion protein.

The above-mentioned protease cleavage sequence(s) may be introduced (and/or any inherent cleavage sequence removed) at the DNA level by conventional means, such as by site-directed mutagenesis. Screening to confirm the presence of cleavage sequences may be performed manually or with the assistance of computer software (e.g. the MapDraw program by DNASTAR, Inc.). Whilst any protease cleavage site may be employed (i.e., clostridial, or non-clostridial), the following are preferred:

Enterokinase (DDDDK↓) (SEQ ID NO: 59) Factor Xa (IEGR↓/IDGR↓) (SEQ ID NOs: 60/61) TEV (Tobacco (ENLYFQ↓G) (SEQ ID NO: 62) Etch virus) Thrombin (LVPR↓GS) (SEQ ID NO: 63) PreScission (LEVLFQ↓GP) (SEQ ID NO: 64).

Additional protease cleavage sites include recognition sequences that are cleaved by a non-cytotoxic protease, for example by a clostridial neurotoxin. These include the SNARE (eg. SNAP-25, syntaxin, VAMP) protein recognition sequences that are cleaved by non-cytotoxic proteases such as clostridial neurotoxins. Particular examples are provided in US2007/0166332, which is hereby incorporated in its entirety by reference thereto.

Also embraced by the term protease cleavage site is an intein, which is a self-cleaving sequence. The self-splicing reaction is controllable, for example by varying the concentration of reducing agent present. The above-mentioned ‘activation’ cleavage sites may also be employed as a ‘destructive’ cleavage site (discussed below) should one be incorporated into a polypeptide of the present invention.

In a preferred embodiment, the fusion protein of the present invention may comprise one or more N-terminal and/or C-terminal located purification tags. Whilst any purification tag may be employed, the following are preferred:

His-tag (e.g. 6× histidine), preferably as a C-terminal and/or N-terminal tag

MBP-tag (maltose binding protein), preferably as an N-terminal tag

GST-tag (glutathione-S-transferase), preferably as an N-terminal tag

His-MBP-tag, preferably as an N-terminal tag

GST-MBP-tag, preferably as an N-terminal tag

Thioredoxin-tag, preferably as an N-terminal tag

CBD-tag (Chitin Binding Domain), preferably as an N-terminal tag.

One or more peptide spacer/linker molecules may be included in the fusion protein. For example, a peptide spacer may be employed between a purification tag and the rest of the fusion protein molecule.

Thus, a third aspect of the present invention provides a nucleic acid (e.g. DNA) sequence encoding a polypeptide as described above (i.e. the second aspect of the present invention).

Said nucleic acid may be included in the form of a vector, such as a plasmid, which may optionally include one or more of an origin of replication, a nucleic acid integration site, a promoter, a terminator, and a ribosome binding site.

The present invention also includes a method for expressing the above-described nucleic acid sequence (i.e. the third aspect of the present invention) in a host cell, in particular in E. coli.

The present invention also includes a method for activating a polypeptide of the present invention, said method comprising contacting the polypeptide with a protease that cleaves the polypeptide at a recognition site (cleavage site) located between the non-cytotoxic protease component and the translocation component, thereby converting the polypeptide into a di-chain polypeptide wherein the non-cytotoxic protease and translocation components are joined together by a disulphide bond. In a preferred embodiment, the recognition site is not native to a naturally-occurring clostridial neurotoxin and/or to a naturally-occurring IgA protease.

The polypeptides of the present invention may be further modified to reduce or prevent unwanted side-effects associated with dispersal into non-targeted areas. According to this embodiment, the polypeptide comprises a destructive cleavage site. The destructive cleavage site is distinct from the ‘activation’ site (i.e. di-chain formation), and is cleavable by a second protease and not by the non-cytotoxic protease. Moreover, when so cleaved at the destructive cleavage site by the second protease, the polypeptide has reduced potency (e.g. reduced binding ability to the intended target cell, reduced translocation activity and/or reduced non-cytotoxic protease activity). For completeness, any of the ‘destructive’ cleavage sites of the present invention may be separately employed as an ‘activation’ site in a polypeptide of the present invention.

Thus, according to this embodiment, the present invention provides a polypeptide that can be controllably inactivated and/or destroyed at an off-site location.

In a preferred embodiment, the destructive cleavage site is recognised and cleaved by a second protease (i.e. a destructive protease) selected from a circulating protease (e.g. an extracellular protease, such as a serum protease or a protease of the blood clotting cascade), a tissue-associated protease (e.g. a matrix metalloprotease (MMP), such as an MMP of muscle), and an intracellular protease (preferably a protease that is absent from the target cell).

Thus, in use, should a polypeptide of the present invention become dispersed away from its intended target cell and/or be taken up by a non-target cell, the polypeptide will become inactivated by cleavage of the destructive cleavage site (by the second protease).

In one embodiment, the destructive cleavage site is recognised and cleaved by a second protease that is present within an off-site cell-type. In this embodiment, the off-site cell and the target cell are preferably different cell types. Alternatively (or in addition), the destructive cleavage site is recognised and cleaved by a second protease that is present at an off-site location (e.g. distal to the target cell). Accordingly, when destructive cleavage occurs extracellularly, the target cell and the off-site cell may be either the same or different cell-types. In this regard, the target cell and the off-site cell may each possess a receptor to which the same polypeptide of the invention binds.

The destructive cleavage site of the present invention provides for inactivation/destruction of the polypeptide when the polypeptide is in or at an off-site location. In this regard, cleavage at the destructive cleavage site minimises the potency of the polypeptide (when compared with an identical polypeptide lacking the same destructive cleavage site, or possessing the same destructive site but in an uncleaved form). By way of example, reduced potency includes: reduced binding (to a mammalian cell receptor) and/or reduced translocation (across the endosomal membrane of a mammalian cell in the direction of the cytosol), and/or reduced SNARE protein cleavage.

When selecting destructive cleavage site(s) in the context of the present invention, it is preferred that the destructive cleavage site(s) are not substrates for any proteases that may be separately used for post-translational modification of the polypeptide of the present invention as part of its manufacturing process. In this regard, the non-cytotoxic proteases of the present invention typically employ a protease activation event (via a separate ‘activation’ protease cleavage site, which is structurally distinct from the destructive cleavage site of the present invention). The purpose of the activation cleavage site is to cleave a peptide bond between the non-cytotoxic protease and the translocation or the binding components of the polypeptide of the present invention, thereby providing an ‘activated’ di-chain polypeptide wherein said two components are linked together via a di-sulfide bond.

Thus, to help ensure that the destructive cleavage site(s) of the polypeptides of the present invention do not adversely affect the ‘activation’ cleavage site and subsequent di-sulfide bond formation, the former are preferably introduced into polypeptide of the present invention at a position of at least 20, at least 30, at least 40, at least 50, and more preferably at least 60, at least 70, at least 80 (contiguous) amino acid residues away from the ‘activation’ cleavage site.

The destructive cleavage site(s) and the activation cleavage site are preferably exogenous (i.e. engineered/artificial) with regard to the native components of the polypeptide. In other words, said cleavage sites are preferably not inherent to the corresponding native components of the polypeptide. By way of example, a protease or translocation component based on BoNT/A L-chain or H-chain (respectively) may be engineered according to the present invention to include a cleavage site. Said cleavage site would not, however, be present in the corresponding BoNT native L-chain or H-chain. Similarly, when the Targeting Moiety component of the polypeptide is engineered to include a protease cleavage site, said cleavage site would not be present in the corresponding native sequence of the corresponding Targeting Moiety.

In a preferred embodiment of the present invention, the destructive cleavage site(s) and the ‘activation’ cleavage site are not cleaved by the same protease. In one embodiment, the two cleavage sites differ from one another in that at least one, more preferably at least two, particularly preferably at least three, and most preferably at least four of the tolerated amino acids within the respective recognition sequences is/are different.

By way of example, in the case of a polypeptide chimera containing a Factor Xa ‘activation’ site between clostridial L-chain and H_(N) components, it is preferred to employ a destructive cleavage site that is a site other than a Factor Xa site, which may be inserted elsewhere in the L-chain and/or H_(N) and/or TM component(s). In this scenario, the polypeptide may be modified to accommodate an alternative ‘activation’ site between the L-chain and H_(N) components (for example, an enterokinase cleavage site), in which case a separate Factor Xa cleavage site may be incorporated elsewhere into the polypeptide as the destructive cleavage site. Alternatively, the existing Factor Xa ‘activation’ site between the L-chain and H_(N) components may be retained, and an alternative cleavage site such as a thrombin cleavage site incorporated as the destructive cleavage site.

When identifying suitable sites within the primary sequence of any of the components of the present invention for inclusion of cleavage site(s), it is preferable to select a primary sequence that closely matches with the proposed cleavage site that is to be inserted. By doing so, minimal structural changes are introduced into the polypeptide. By way of example, cleavage sites typically comprise at least 3 contiguous amino acid residues. Thus, in a preferred embodiment, a cleavage site is selected that already possesses (in the correct position(s)) at least one, preferably at least two of the amino acid residues that are required in order to introduce the new cleavage site. By way of example, in one embodiment, the Caspase 3 cleavage site (DMQD (SEQ ID NO: 65) may be introduced. In this regard, a preferred insertion position is identified that already includes a primary sequence selected from, for example, Dxxx, xMxx, xxQx, xxxD, DMxx, DxQx, DxxD, xMQx, xMxD, xxQD, DMQx, xMQD, DxQD, and DMxD.

Similarly, it is preferred to introduce the cleavage sites into surface exposed regions. Within surface exposed regions, existing loop regions are preferred.

In a preferred embodiment of the present invention, the destructive cleavage site(s) are introduced at one or more of the following position(s), which are based on the primary amino acid sequence of BoNT/A. Whilst the insertion positions are identified (for convenience) by reference to BoNT/A, the primary amino acid sequences of alternative protease domains and/or translocation domains may be readily aligned with said BoNT/A positions.

For the protease component, one or more of the following positions is preferred: 27-31, 56-63, 73-75, 78-81, 99-105, 120-124, 137-144, 161-165, 169-173, 187-194, 202-214, 237-241, 243-250, 300-304, 323-335, 375-382, 391-400, and 413-423. The above numbering preferably starts from the N-terminus of the protease component of the present invention.

In a preferred embodiment, the destructive cleavage site(s) are located at a position greater than 8 amino acid residues, preferably greater than 10 amino acid residues, more preferably greater than 25 amino acid residues, particularly preferably greater than 50 amino acid residues from the N-terminus of the protease component. Similarly, in a preferred embodiment, the destructive cleavage site(s) are located at a position greater than 20 amino acid residues, preferably greater than 30 amino acid residues, more preferably greater than 40 amino acid residues, particularly preferably greater than 50 amino acid residues from the C-terminus of the protease component.

For the translocation component, one or more of the following positions is preferred: 474-479, 483-495, 507-543, 557-567, 576-580, 618-631, 643-650, 669-677, 751-767, 823-834, 845-859. The above numbering preferably acknowledges a starting position of 449 for the N-terminus of the translocation domain component of the present invention, and an ending position of 871 for the C-terminus of the translocation domain component.

In a preferred embodiment, the destructive cleavage site(s) are located at a position greater than 10 amino acid residues, preferably greater than 25 amino acid residues, more preferably greater than 40 amino acid residues, particularly preferably greater than 50 amino acid residues from the N-terminus of the translocation component. Similarly, in a preferred embodiment, the destructive cleavage site(s) are located at a position greater than 10 amino acid residues, preferably greater than 25 amino acid residues, more preferably greater than 40 amino acid residues, particularly preferably greater than 50 amino acid residues from the C-terminus of the translocation component.

In a preferred embodiment, the destructive cleavage site(s) are located at a position greater than 10 amino acid residues, preferably greater than 25 amino acid residues, more preferably greater than 40 amino acid residues, particularly preferably greater than 50 amino acid residues from the N-terminus of the TM component. Similarly, in a preferred embodiment, the destructive cleavage site(s) are located at a position greater than 10 amino acid residues, preferably greater than 25 amino acid residues, more preferably greater than 40 amino acid residues, particularly preferably greater than 50 amino acid residues from the C-terminus of the TM component.

The polypeptide of the present invention may include one or more (e.g. two, three, four, five or more) destructive protease cleavage sites. Where more than one destructive cleavage site is included, each cleavage site may be the same or different. In this regard, use of more than one destructive cleavage site provides improved off-site inactivation. Similarly, use of two or more different destructive cleavage sites provides additional design flexibility.

The destructive cleavage site(s) may be engineered into any of the following component(s) of the polypeptide: the non-cytotoxic protease component; the translocation component; the Targeting Moiety; or the spacer peptide (if present). In this regard, the destructive cleavage site(s) are chosen to ensure minimal adverse effect on the potency of the polypeptide (for example by having minimal effect on the targeting/binding regions and/or translocation domain, and/or on the non-cytotoxic protease domain) whilst ensuring that the polypeptide is labile away from its target site/target cell.

Preferred destructive cleavage sites (plus the corresponding second proteases) are listed in the Table immediately below. The listed cleavage sites are purely illustrative and are not intended to be limiting to the present invention.

Destructive cleavage site Tolerated recognition sequence variance Second recognition P4-P3-P2-P1-▾-P1′-P2′-P3′ protease sequence P4 P3 P2 P1 P1′ P2′ P3′ Thrombin LVPR▾GS A, F, G, I, A, F, G, P R Not D Not — (SEQ ID NO: 63) L, T, V I, L, T, or E D or E or M V, W or A Thrombin GR▾G G R G Factor Xa IEGR▾ A, F, G, I, D or E G R — — — (SEQ ID NO: 60) L, T, V or M ADAM17 PLAQA▾VRSSS (SEQ ID NO: 66) Human SKGR▾SLIGRV airway (SEQ ID NO: 67) trypsin-like protease (HAT) ACE — — — — Not P Not N/A (peptidyl- D or E dipeptidase A) Elastase MEA▾VTY M, R E A, H V, T V, T, H Y — (leukocyte) (SEQ ID NO: 68) Furin RXR/KR▾ R X R R (SEQ ID NO: 69) or K Granzyme IEPD▾ I E P D — — — (SEQ ID NO: 70) Caspase 1 F, W, Y, L — H, D Not — — A, T P, E.D. Q.K or R Caspase 2 DVAD▾ D V A D Not — — (SEQ ID NO: 71) P, E.D. Q.K or R Caspase 3 DMQD▾ D M Q D Not — — (SEQ ID NO: 72) P, E.D. Q.K or R Caspase 4 LEVD▾ L E V D Not — — (SEQ ID NO: 73) P, E.D. Q.K or R Caspase 5 L or W E H D — — — Caspase 6 V E H D Not — — or I P, E.D. Q.K or R Caspase 7 DEVD▾ D E V D Not — — (SEQ ID NO: 74) P, E.D. Q.K or R Caspase 8 I or L E T D Not — — P, E.D. Q.K or R Caspase 9 LEHD▾ L E H D — — — (SEQ ID NO: 75) Caspase IEHD▾ I E H D — — — 10 (SEQ ID NO: 76)

Matrix metalloproteases (MMPs) are a preferred group of destructive proteases in the context of the present invention. Within this group, ADAM17 (EC 3.4.24.86, also known as TACE), is preferred and cleaves a variety of membrane-anchored, cell-surface proteins to “shed” the extracellular domains. Additional, preferred MMPs include adamalysins, serralysins, and astacins.

Another group of preferred destructive proteases is a mammalian blood protease, such as Thrombin, Coagulation Factor Vila, Coagulation Factor IXa, Coagulation Factor Xa, Coagulation Factor XIa, Coagulation Factor XIIa, Kallikrein, Protein C, and MBP-associated serine protease.

In one embodiment of the present invention, said destructive cleavage site comprises a recognition sequence having at least 3 or 4, preferably 5 or 6, more preferably 6 or 7, and particularly preferably at least 8 contiguous amino acid residues. In this regard, the longer (in terms of contiguous amino acid residues) the recognition sequence, the less likely non-specific cleavage of the destructive site will occur via an unintended second protease.

It is preferred that the destructive cleavage site of the present invention is introduced into the protease component and/or the Targeting Moiety and/or into the translocation component and/or into the spacer peptide. Of these four components, the protease component is preferred. Accordingly, the polypeptide may be rapidly inactivated by direct destruction of the non-cytotoxic protease and/or binding and/or translocation components.

Polypeptide Delivery

In use, the present invention employs a pharmaceutical composition, comprising a polypeptide, together with at least one component selected from a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/or salt.

The polypeptides of the present invention may be formulated for oral, parenteral, continuous infusion, implant, inhalation or topical application. Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use.

Local delivery means may include an aerosol, or other spray (eg. a nebuliser). In this regard, an aerosol formulation of a polypeptide enables delivery to the lungs and/or other nasal and/or bronchial or airway passages.

The preferred route of administration is selected from: systemic, laparoscopic and/or localised injection (e.g. injection directly into the tumour).

In the case of formulations for injection, it is optional to include a pharmaceutically active substance to assist retention at or reduce removal of the polypeptide from the site of administration. One example of such a pharmaceutically active substance is a vasoconstrictor such as adrenaline. Such a formulation confers the advantage of increasing the residence time of polypeptide following administration and thus increasing and/or enhancing its effect.

The dosage ranges for administration of the polypeptides of the present invention are those to produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the polypeptide or composition, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.

Suitable daily dosages (per kg weight of patient) are in the range 0.0001-1 mg/kg, preferably 0.0001-0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and particularly preferably 0.004-0.5 mg/kg. The unit dosage can vary from less that 1 microgram to 30 mg, but typically will be in the region of 0.01 to 1 mg per dose, which may be administered daily or preferably less frequently, such as weekly or six monthly.

A particularly preferred dosing regimen is based on 2.5 ng of polypeptide as the 1× dose. In this regard, preferred dosages are in the range 1×-100× (i.e. 2.5-250 ng).

Fluid dosage forms are typically prepared utilising the polypeptide and a pyrogen-free sterile vehicle. The polypeptide, depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle. In preparing solutions the polypeptide can be dissolved in the vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving. Advantageously additives such as buffering, solubilising, stabilising, preservative or bactericidal, suspending or emulsifying agents and or local anaesthetic agents may be dissolved in the vehicle.

Dry powders, which are dissolved or suspended in a suitable vehicle prior to use, may be prepared by filling pre-sterilised ingredients into a sterile container using aseptic technique in a sterile area. Alternatively the ingredients may be dissolved into suitable containers using aseptic technique in a sterile area. The product is then freeze dried and the containers are sealed aseptically.

Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration. The components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation.

Advantageously, a suspending agent for example polyvinylpyrrolidone is included in the composition/s to facilitate uniform distribution of the components.

Administration in accordance with the present invention may take advantage of a variety of delivery technologies including microparticle encapsulation, viral delivery systems or high-pressure aerosol impingement.

Definitions Section

Targeting Moiety (TM) means any chemical structure that functionally interacts with a Binding Site to cause a physical association between the polypeptide of the invention and the surface of a target cell. In the context of the present invention, the target cell is a cancerous cell, for example one in which autocrine stimulation is driving proliferation, survival, metastatic potential or angiogenesis. The term TM embraces any molecule (ie. a naturally occurring molecule, or a chemically/physically modified variant thereof) that is capable of binding to a Binding Site on the target cell, which Binding Site is capable of internalisation (eg. endosome formation)—also referred to as receptor-mediated endocytosis. The TM may possess an endosomal membrane translocation function, in which case separate TM and Translocation Domain components need not be present in an agent of the present invention. Throughout the preceding description, specific TMs have been described. Reference to said TMs is merely exemplary, and the present invention embraces all variants and derivatives thereof, which retain the basic binding (i.e. targeting) ability of the exemplified TMs.

In one embodiment, the cancer cell target of the present invention may be a cancer cell that does not secrete growth hormone (ie. no significant or detectable growth hormone is secreted therefrom).

As mentioned previously, preferred TMs include antibodies (eg. antibody fragments) and binding scaffolds; especially commercially available antibodies/fragments and scaffolds designed for the purpose of binding (eg. specifically) to cancer cells.

Protein scaffolds represent a new generation of universal binding frameworks to complement the expanding repertoire of therapeutic monoclonal antibodies and derivatives such as scFvs, Fab molecules, dAbs (single-domain antibodies), diabodies and minibodies, each of which may be employed as a TM of the present invention. Scaffold systems create or modify known protein recognition domains either through creation of novel scaffolds or modification of known protein binding domains. Such scaffolds include but are not limited to:

(i) protein A based scaffolds-affibodies (Nord, K. et al 1997 “Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain”. Nat Biotechnol 15, 772-777);

(ii) lipocalin based scaffolds—anticalins (Skerra 2008 “Alternative binding proteins: anticalins—harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities”. FEBS J. 275:2677-83);

(iii) fibronectin based scaffolds—adnectin (Dineen et al 2008 “The Adnectin CT-322 is a novel VEGF receptor 2 inhibitor that decreases tumor burden in an orthotopic mouse model of pancreatic cancer”. BMC Cancer 8:352);

(iv) avimers (Silverman et al 2005 “Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains”. Nat Biotechnol 23:1556-61);

(v) ankyrin based scaffolds—darpins (Zahnd et al 2006 “Selection and characterization of Her2 binding-designed ankyrin repeat proteins”. J Biol Chem. 281:35167-75); and

(vi) centyrin scaffolds—based on a protein fold that has significant structural homology to Ig domains with loops that are analogous to CDRs. Ig domains are a common module in human proteins and have been widely applied as alternative scaffold proteins. Each of the above ‘scaffold’ publications is hereby incorporated (in its entirety) by reference thereto.

Binding scaffolds can be used to target particular cell types via interaction with specific cell surface proteins, receptors or other cell surface epitopes such as sugar groups. Such modified scaffolds can be engineered onto recombinant non-cytotoxic protease based polypeptides of the present invention to target specific cancer cell types of interest.

The TM of the present invention binds (preferably specifically binds) to the target cell in question. The term “specifically binds” preferably means that a given TM binds to the target cell with a binding affinity (Ka) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater, more preferably 10⁸ M⁻¹ or greater, and most preferably, 10⁹ M⁻¹ or greater.

Reference to TM in the present specification embraces fragments and variants thereof, which retain the ability to bind to the target cell in question. By way of example, a variant may have at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 97 or at least 99% amino acid sequence homology with the reference TM. Thus, a variant may include one or more analogues of an amino acid (e.g. an unnatural amino acid), or a substituted linkage. Also, by way of example, the term fragment, when used in relation to a TM, means a peptide having at least ten, preferably at least twenty, more preferably at least thirty, and most preferably at least forty amino acid residues of the reference TM. The term fragment also relates to the above-mentioned variants. Thus, by way of example, a fragment of the present invention may comprise a peptide sequence having at least 10, 20, 30 or 40 amino acids, wherein the peptide sequence has at least 80% sequence homology over a corresponding peptide sequence (of contiguous) amino acids of the reference peptide.

By way of example, ErbB peptide TMs may be modified to generate mutein ErbB ligands with improved properties such as increased stability. By way of example, ErbB TM muteins include ErbB peptides having amino acid modifications such as a valine residue at position 46 or 47 (EGFVal46 or 47), which confers stability to cellular proteases. ErbB TMs may also have amino acids deleted or additional amino acids inserted. This includes but is not limited to EGF having a deletion of the two C-terminal amino acids and a neutral amino acid substitution at position 51 (particularly EGF51Gln51; see US20020098178A1), and EGF with amino acids deleted (e.g. rEGF2-48; rEGF3-48 and rEGF4-48). Fragments of ErbB TMs may include fragments of TGFα which contain predicted β-turn regions (e.g. a peptide of the sequence Ac-C-H-S-G-Y-V-G-A-R-C-O-OMe) (SEQ ID NO: 77), fragments of EGF such as [Ala20]EGF(14-31), and the peptide YHWYGYTPQNVI (SEQ ID NO: 78) or GE11. All of the above patent specifications are incorporated herein by reference thereto.

By way of further example, somatostatin (SST) and cortistatin (CST) have high structural homology, and bind to all known SST receptors. Full-length SST has the amino acid sequence:

(SEQ ID NO: 79) MLSCRLQCALAALSIVLALGCVTGAPSDPRLRQFLQKSLAAAAGKQELAK YFLAELLSEPNQTENDALEPEDLSQAAEQDEMRLELQRSANSNPAMAPRE RKAGCKNFFWKTFTSC.

Full-length CST has the amino acid sequence:

(SEQ ID NO: 80) MYRHKNSWRLGLKYPPSSKEETQVPKTLISGLPGRKSSSRVGEKLQSAHK MPLSPGLLLLLLSGATATAALPLEGGPTGRDSEHMQEAAGIRKSSLLTFL AWWFEWTSQASAGPLIGEEAREVARRQEGAPPQQSARRDRMPCRNFFWKT FSSCK.

Reference to these TMs includes the following fragments (and corresponding variants) thereof:

(SEQ ID NO: 81)                  NFFWKTF; (SEQ ID NO: 82)          (R or K)NFFWKTF; (SEQ ID NO: 83)         C(R or K)NFFWKTF; (SEQ ID NO: 84) (P or G)C(R or K)NFFWKTF; (SEQ ID NO: 85)                  NFFWKTF(S or T); (SEQ ID NO: 86)                  NFFWKTF(S or T)S; (SEQ ID NO: 87)                  NFFWKTF(S or T)SC; (SEQ ID NO: 88)          (R or K)NFFWKTF(S or T); (SEQ ID NO: 89)          (R or K)NFFWKTF(S or T)S; (SEQ ID NO: 90)          (R or K)NFFWKTF(S or T)SC; (SEQ ID NO: 91)         C(R or K)NFFWKTF(S or T); (SEQ ID NO: 92)         C(R or K)NFFWKTF(S or T)S; (SEQ ID NO: 93)         C(R or K)NFFWKTF(S or T)SC; (SEQ ID NO: 94) (P or G)C(R or K)NFFWKTF(S or T); (SEQ ID NO: 95) (P or G)C(R or K)NFFWKTF(S or T)S; or (SEQ ID NO: 96) (P or G)C(R or K)NFFWKTF(S or T)C.

With regard to the above sequences, where a (P or G) alternative is given, a P is preferred in the case of a CST TM, whereas a G is preferred in the case of an SST TM. Where an (R or K) alternative is given, an R is preferred in the case of a CST TM, whereas a K is preferred in the case of an SST TM. Where an (S or T) alternative is given, an S is preferred in the case of a CST TM, whereas a T is preferred in the case of an SST TM.

Preferred fragments comprise at least 7 or at least 10 amino acid residues, preferably at least 14 or at least 17 amino acid residues, and more preferably at least 28 or 29 amino acid residues. By way of example, preferred sequences include:

(SEQ ID NO: 97) SANSNPAMAPRERKAGCKNFFWKTFTSC (SST-28); (SEQ ID NO: 98) AGCKNFFWKTFTSC (SST-14); (SEQ ID NO: 99) QEGAPPQQSARRDRMPCRNFFWKTFSSCK (CST-29); (SEQ ID NO: 100) QERPPLQQPPHRDKKPCKNFFWKTFSSCK (CST-29); (SEQ ID NO: 101) QERPPPQQPPHLDKKPCKNFFWKTFSSCK (CST-29); (SEQ ID NO: 102) DRMPCRNFFWKTFSSCK (CST-17); (SEQ ID NO: 103) PCRNFFWKTFSSCK (CST-14); and (SEQ ID NO: 104) PCKNFFWKTFSSCK (CST-14).

The TM may comprise a longer amino acid sequence, for example, at least 30 or 35 amino acid residues, or at least 40 or 45 amino acid residues, so long as the TM is able to bind to a normal GH-secreting cell, preferably to an SST or to a CST receptor on a normal GH-secreting cell. In this regard, the TM is preferably a fragment of full-length SST or CST, though including at least the core sequence “NFFWKTF” or one of the above-defined primary amino acid sequences.

It is routine to confirm that a TM binds to the selected target cell. For example, a simple radioactive displacement experiment may be employed in which tissue or cells representative of a growth hormone-secreting cell are exposed to labelled (eg. tritiated) TM in the presence of an excess of unlabelled TM. In such an experiment, the relative proportions of non-specific and specific binding may be assessed, thereby allowing confirmation that the TM binds to the target cell. Optionally, the assay may include one or more binding antagonists, and the assay may further comprise observing a loss of TM binding. Examples of this type of experiment can be found in Hulme, E. C. (1990), Receptor-binding studies, a brief outline, pp. 303-311, In Receptor biochemistry, A Practical Approach, Ed. E. C. Hulme, Oxford University Press.

In the context of the present invention, reference to a peptide TM (e.g. GHRH peptide, or leptin peptide) embraces peptide analogues thereof, so long as the analogue binds to the same receptor as the corresponding ‘reference’ TM. Said analogues may include synthetic residues such as:

β-Nal=β-naphthylalanine β-Pal=β-pyridylalanine hArg(Bu)=N-guanidino-(butyl)-homoarginine hArg(Et)₂=N, N′-guanidino-(dimethyl)-homoarginine hArg(CH₂CF₃)₂=N, N-guanidino-bis-(2,2,2,-trifluoroethyl)-homoarginine hArg(CH₃, hexyl)=N, N-guanidino-(methyl, hexyl)-homoarginine

Lys(Me)=N^(e)-methyllysine Lys(iPr)=N^(e)-isopropyllysine

AmPhe=aminomethylphenylalanine AChxAla=aminocyclohexylalanine Abu=α-aminobutyric acid Tpo=4-thiaproline

MeLeu=N-methylleucine

Orn=omithine Nle=norleucine Nva=norvaline Trp(Br)=5-bromo-tryptophan Trp(F)=5-fluoro-tryptophan Trp(N0₂)=5-nitro-tryptophan Gaba=γ-aminobutyric acid

Bmp=J-mercaptopropionyl

Ac=acetyl Pen—pencillamine

By way of example, the above peptide analogue aspect is described in more detail with reference to specific peptide TMs, such as SST peptides, GHRH peptides, bombesin peptides, GnRH peptides, and ghrelin peptides, though the same principle applies to all TMs of the present invention.

Somatostatin analogues, which can be used to practice the present invention include, but are not limited to, those described in the following publications, which are hereby incorporated by reference: Van Binst, G. et al. Peptide Research 5: 8 (1992); Horvath, A. et al. Abstract, “Conformations of Somatostatin Analogs Having Antitumor Activity”, 22nd European peptide Symposium, Sep. 13-19,1992, Interlaken, Switzerland; U.S. Pat. No. 5,506,339; EP0363589; U.S. Pat. Nos. 4,904,642; 4,871,717; 4,725,577; 4,684,620; 4,650,787; 4,585,755; 4,725,577; 4,522,813; 4,369,179; US4.360,516: U.S. Pat. No. 4,328,214; US4,316.890; US4,310.518; US4,291.022; U.S. Pat. Nos. 4,238,481; 4,235,886; 4,211,693; 4,190,648; 4,146,612; 4,133,782; US5.506,339: U.S. Pat. Nos. 4,261,885; 4,282,143; 4,190,575; 5,552,520; EP0389180; EP0505680; U.S. Pat. No. 4,603,120; EP0030920; U.S. Pat. No. 4,853,371; WO90/12811; WO97/01579; WO91/18016; WO98/08529 and WO98/08528; WO/0075186 and WO00/06185; WO099/56769; and FR 2,522,655. Each of these publications is incorporated in its entirety by reference thereto.

Methods for synthesizing analogues are well documented, as illustrated, for example, by the patents cited above. For example, synthesis of H-D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2, can be achieved by following the protocol set forth in Example 1 of EP0395417A1. Similarly, synthesis analogues with a substituted N-terminus can be achieved, for example, by following the protocol set forth in WO88/02756, EP0329295, and U.S. Pat. No. 5,240,561.

The use of linear SST analogues are also included within the scope of this invention, for example: H-D-Phe-p-chloro-Phe-Tyr-D-Trp-Lys-Thr-Phe-Thr-NH2; H-D-Phe-p-N02-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-N H2; H-D-*Nal-p-chloro-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH2; H-D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2; H-D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH2; H-D-Phe-p-chloro-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH2; and H-D-Phe-Ala-Tyr-D-Trp-Lys-Val-Ala-D-beta-Nal-NH2.

One or more chemical moieties, eg. a sugar derivative, mono or poly-hydroxy (C2-12) alkyl. mono or poly-hydroxy (C2-12) acyl groups. or a piperazine derivative, can be attached to a SST analogue, e g. to the N-terminus amino acid—see WO88/02756, EP0329295, and U.S. Pat. No. 5,240,561.

GHRH peptide analogues date back to the 1990s, and include the ‘standard antogonist’ [Ac-Tyr′, D-Arg2jhGH-RH (1-29) Nha. U.S. Pat. No. 4,659,693 discloses GH-RH antagonistic analogs which contain certain N, N′-dialkyl-omega-guanidino alpha-amino acyl residues in position 2 of the GH-RH (1-29) sequence. Additional examples are provided in WO91/16923, U.S. Pat. No. 5,550,212, 5,942,489, 6,057,422 5,942,489, 6,057,422, WO96/032126, WO96/022782, WO96/016707, WO94/011397, WO94/011396, each of which is herein incorporated by reference thereto.

Examples of bombesin analogues suitable for use in the present invention include TMs comprising: D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (code named BIM-26218), D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH₂ (code named BIM-26187); D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu-q [CH₂NH]-Phe-NH₂ (code named BIM-26159), and D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-q [CH₂NH]-Cpa-NH₂ (code named BIM-26189); D-Phe-Gln-Trp-Ala-Val-N-methyl-D-Ala-His-Leu- methylester, and D-F_(g)-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-methylester.

Bombesin analogues include peptides derived from the naturally-occurring. structurally-related peptides, namely, bombesin, neuromedin B, neuromedin C, litorin, and GRP. The relevant amino acid sequences of these naturally occurring TM peptides are listed below:

Bombesin (last 10 aa's): (SEQ ID NO: 105) Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂; Neuromedin B: (SEQ ID NO: 106) Gly-Asn-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH₂; Neuromedin C: (SEQ ID NO: 107) Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH₂; Litorin: (SEQ ID NO: 108) pGlu-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH₂; Human GRP (last 10 aa's): (SEQ ID NO: 109) Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH₂.

Analogs suitable for use in the present invention are described in U.S. Serial Number 502,438, filed Mar. 30, 1990, U.S. Serial No. 397,169, filed Aug. 21, 1989, U.S. Serial No. 376,555, filed Jul. 7, 1989, U.S. Serial Number 394,727, filed Aug. 16, 1989, U.S. Serial No. 317,941, filed Mar. 2, 1989, U.S. Serial Number 282,328, filed Dec. 9, 1988, U.S. Serial No. 257,998, filed Oct. 14, 1988, U.S. Serial No. 248,771, filed Sep. 23, 1988, U.S. Serial No. 207,759, filed Jun. 16, 1988, U.S. Serial No. 204,171, filed Jun. 8, 1988. U.S. Ser. No. 173,311, filed Mar. 25, 1988, U.S. Ser. No. 100,571, filed Sep. 24, 1987; and U.S. Serial No. 520,225, filed May 9, 1990, U.S. Serial No. 440,039, filed Nov. 21, 1989. All these applications are hereby incorporated by reference. Bombesin analogs are also described in Zachary et al., Proc. Nat. Aca. Sci. 82:7616 (1985); Heimbrook et al., “Synthetic Peptides: Approaches to Biological Problems”, UCLA Symposium on Mol. and Cell. Biol. New Series. Vol. 86. ed. Tarn and Kaiser; Heinz-Erian et al., Am. J. Physiol. G439 (1986); Martinez et al., J. Med. Chem. 28:1874 (1985); Gargosky et al., Biochem. J. 247:427 (1987); Dubreuil et al., Drug Design and Delivery, Vol 2:49. Harwood Academic Publishers, GB (1987): Heikkila et al., J. Biol. Chem. 262:16456 (1987); Caranikas et al., J. Med. Chem. 25:1313 (1982); Saeed et al., Peptides 10:597 (1989); Rosell et al., Trends in Pharmacological Sciences 3:211 (1982); Lundberg et al., Proc. Nat. Aca. Sci. 80:1120. (1983); Engberg et al., Nature 293:222 (1984); Mizrahi et al., Euro. J. Pharma. 82:101 (1982); Leander et al., Nature 294:467 (1981); Woll et al., Biochem. Biophys. Res. Comm. 155:359 (1988); Rivier et al., Biochem. 17:1766 (1978); Cuttitta et al., Cancer Surveys 4:707 (1985); Aumelas et al., Int. J. Peptide Res. 30:596 (1987): all of which are also hereby incorporated by reference.

The analogs can be prepared by conventional techniques, such as those described in WO92/20363 and EP0737691. Additional bombesin analogues are described in, for example, WO89/02897, WO91/17181, WO90/03980 and WO91/02746, all of which are herein incorporated by reference thereto.

Examples of ghrelin analogues suitable for use as a TM of the present invention comprise: Tyr-DTrp-DLys-Trp-DPhe-NH₂, Tyr-DTrp-Lys-Trp-DPhe-NH₂, His-DTrp-DLys-Trp-DPhe-NH₂, His-DTrp-DLys-Phe-DTrp-NH₂, His-DTrp-DArg-Trp-DPhe-NH₂, His-DTrp-DLys-Trp-DPhe-Lys-NH₂, DesaminoTyr-DTrp-Ala-Trp-DPhe-NH₂, DesaminoTyr-DTrp-DLys-Trp-DPhe-NH₂, DeaminoTyr-DTrp-Ser-Trp-DPhe-Lys-NH₂, DesaminoTyr-DTrp-Ser-Trp-DPhe-NH₂, His-DTrp-DTrp-Phe-Met-NH₂, Tyr-DTrp-DTrp-Phe-Phe-NH₂, Glyψ[CH₂NH]-DβNal-Ala-Trp-DPhe-Lys-NH₂, Glyψ[CH2NH]-DbetaNal-DLyS-TrP-DPhe-Lys-NH₂, DAla-DbetaNal-DLys-DTrp-Phe-Lys-NH₂, His-DbetaNal-DLys-Trp-DPhe-Lys-NH₂, Ala-His-DTrp-DLys-Trp-DPhe-Lys-NH₂, Alaφ[CH₂NH]-DbetaNal-Ala-Trp-DPhe-Lys-NH₂, DbetaNal-Ala-Trp-DPhe-Ala-NH₂, DAla-DcyclohexylAla-Ala-Phe-DPhe-Nle-NH₂, DcyclohexylAla-Ala-Phe-DTrp-Lys-NH₂. DAla-DbetaAla-Thr-DThr-Lys-NH₂, DcyclohexylAla-Ala-Trp-DPhe-NH2, DAla-DbetaNal-Ala-Ala-DAla-Lys-NH₂, DbetaNal-Ala-Trp-DPhe-Leu-NH₂, His-DTrp-Phe-Trp-DPhe-Lys-NH₂, DAla-DbetaNal-DAla-DTrp-Phe-Lys-NH₂, pAla-Trp-DAla-DTrp-Phe-N H₂, His-Trp-DAla-DTrp-Phe-LysNH₂, DLys-DβNal-Ala-Trp-DPhe-Lys-NH₂, DAla-DbetaNal-D Lys-DTrp-Phe-Lys-NH₂, Tyr-DAla-Phe-Aib-NH₂, Tyr-DAla-Sar-NMePhe-NH₂, αγAbu-DTrp-DTrp-Ser-NH₂, αγAbu-DTrp-DTrp-Lys-NH₂, αγAbu-DTrp-DTrp-Orn-N H₂, αAbu-DTrp-DTrp-Orn-N H₂, DThr-DαNal-DTrp-DPro-Arg-NH₂, DAla-Ala-DAla-DTrp-Phe-Lys-NH₂, Alaψ[CH₂NH]His-DTrp-Ala-Trp-DPhe-Lys-NH₂, Lys-DHis-DTrp-Phe-NH₂, γAbu-DTrp-DTrp-Orn-NH₂, inip-Trp-Trp-Phe-NH₂, Ac-DTrp-Phe-DTrp-Leu-NH₂, Ac-DTrp-Phe-DTrp-Lys-N H₂, Ac-DTrp-DTrp-Lys-NH₂, DLys-Tyr-DTrp-DTrp-Phe-Lys-NH₂, Ac-DbetaNal-Leu-Pro-NH₂, pAla-Trp-DTrp-DTrp-Orn-NH₂, DVal-DαNal-DTrp-Phe-Arg-NH₂, DLeu-DαNal-DTrp-Phe-Arg-NH₂, CyclohexylAla-DaNal-DTrp-Phe-Arg-NH₂, DTp-DαNal-DTrp-Phe-Arg-NH₂, DAla-DβNal-DPro-Phe-Arg-NH₂, Ac-DαNal-DTrp-Phe-Arg-NH₂, DαNal-DTrp-Phe-Arg-NH₂, His-DTrp-DTrp-Lys-NH₂, Ac-DpNal-DTrp-NH₂, αAib-DTrp-DcyclohexylAla-NH₂, αAib-DTrp-DAla-cyclohexylAla-NH₂, DAla-DcyclohexylAla-Ala-Ala-Phe-DPhe-Nle-NH₂, DPhe-Ala-Phe-DPal-NH₂, DPhe-Ala-Phe-DPhe-Lys-N H₂, DLys-Tyr-DTrp-DTrp-Phe-NH₂, Ac-DLys-Tyr-DTrp-DTrp-Phe-NH₂, Arg-DTrp-Leu-Tyr-Trp-Pro(cyclic Arg-Pro), Ac-DβNal-PicLys-ILys-DPhe-NH2, DPal-Phe-DTrp-Phe-Met-NH₂, DPhe-Trp-DPhe-Phe-Met-NH₂, DPal-Trp-DPhe-Phe-Met-NH₂, pAla-Pal-DTrp-DTrp-Orn-NH₂, αγAbu-Trp-DTrp-DTrp-Orn-NH₂, βAla-Trp-DTrp-DTrp-Lys-NH₂, γAbu-Trp-DTrp-DTrp-Orn-NH₂, Ava-Trp-DTrp-DTrp-Orn-N H₂, DLys-Tyr-DTrp-Ala-Trp-DPhe-NH₂, His-DTrp-DArg-Trp-DPhe-NH₂, <Glu-His-Trp-DSer-DArg-NH₂, DPhe-DPhe-DTrp-Met-DLys-NH₂, 0-(2-methylallyl) benzophonone oxime, (R)-2-amino-3-(IH-indol-3-yl)-I-(4-phenylpiperidin-1-yl)propan-1-one, N—((R)-1-((R)-1-((S)-3-(IH-indol-3-yl)-1-oxo-1-(4-phenylpiperidin-1-yl)propan-2-ylamino)-6-amino-1-oxohexan-2-ylamino)-3-hydroxy-1-oxopropan-2-yl)benzamide, (S)—N—((S)-3-(1H-indol-3-yl)-1-oxo-1-(4-phenylpiperidin-1-yl)propan-2-yl)-6-acetamido-2-((S)-2-amino-3-(benzyloxy)propanamido)hexanamide, (S)—N—((R)-3-(1H-indol-3-yl)-1-oxo-1-(4-phenylpiperidin-1-yl)propan-2-yl)-2-((S)-2-acetamido-3-(benzyloxy)propanamido)-6-aminohexanamide, (R)—N-(3-(1H-indol-3-yl)-1-(4-(2-methoxyphenyl)piperidin-1-yl)-1-oxopropan-2-yl)-4-aminobutanamide, (R)—N-(3-(1H-indol-3-yl)-1-(4-(2-methoxyphenyl)piperidin-1-yl)-1-oxopropan-2-yl)-2-amino-2-methylpropanamide, methyl 3-(p-tolylcarbamoyl)-2-naphthoate, ethyl 3-(4-(2-methoxyphenyl)piperidine-1-carbonyl)-2-naphthoate, 3-(2-methoxyphenylcarbamoyl)-2-naphthoate, (S)-2,4-diamino-N—((R)-3-(naphthalen-2-ylmethoxy)-1-oxo-1-(4-phenylpiperidin-1-yl)propan-2-yl)butanamide, naphthalene-2,3-diylbis((4-(2-methoxyphenyl)piperazin-1-yl)methanone), (R)-2-amino-N-(3-(benzyloxy)-1-oxo-1-(4-phenylpiperazin-1-yl)propan-2-yl)-2-methylpropanamide, or (R)-2-amino-3-(benzyloxy)-1-(4-phenylpiperazin-1-yl)propan-1-one.

Examples of GnRH analogues suitable for use as a TM in the present invention include those known from, for example, EP171477, WO96/033729, WO92/022322, WO92/013883, and WO91/05563, each of which is herein incorporated by reference thereto. Specific examples comprise: (NAcDQal¹,DPtf²,DPAI³,cjsPzACAla⁵,DPicLys⁶,DAla¹⁰)LHRH; (NAcDNal¹,DpCIPhe²,DPal³,cjsPzACAla⁵,DNicLys⁶,ILys⁸,DAla¹⁰) LHRH; (NAcDNal¹,DpCIPhe²,DPaI³,Thr⁴,PicLys⁵,DPicLys⁶,ILys⁸,DAla¹⁰)LHRH; (NAcDNal¹,DpCIPhe²,DPaI³,PicLys⁵,DPicLys⁶,Thr⁷,ILys⁸,DAla¹⁰)LHRH; (NapDThr¹, DpCIPhe², DPaI³, PicLys⁵,DPicLys⁶,ILys⁸,DAIa¹⁰)LHRH; (NAcDNal¹, DpCIPhe²,DPaI³,NicLys⁵,DNicLys⁶,Thr⁷,ILys⁸,DAla¹⁰)LHRH; (NAcDNal¹, DpCIPhe²,DPaI³,Thr⁴NicLys⁵,DNicLys⁶,Thr⁷,ILys⁸,DAla¹⁰)LHRH; (NAcDNal¹,DpCIPhe²,DPaI³,PicLys⁵,D(PicSar)Lys⁶,ILys⁸,DAla¹⁰)LHRH; (NAcDNal¹,DpCIPhe²,DPaI³,D(PicSar)Lys⁶,ILys⁸,DAla¹⁰)LHRH; (NAcDNal¹,DpCIPhe²,DPaI³,PicLys⁵,D(6ANic)Lys⁶,ILys⁸,DAla¹⁰)LHRH; (NAcDNal¹,DpCIPhe²,DPaI³,PicLys⁵,D(6ANic)0rn⁶,ILys⁸,DAla¹⁰)LHRH; (NAcDQal¹,DCpa²,DPaI³,cisPzACAla⁵,DPicLys⁶,NLeu⁷,ILys⁸,DAla¹⁰)LHRH; (NAcDNal¹,DCpa²,DPal³ DPicLys⁵,DAPhe(PicSar)^(β),ILys⁸,DAla¹⁰)LHRH; (NAcDQal¹,DCpa²,DPaI³,PicLys⁵,DPaI⁶,ILys⁸,DAla¹⁰)LHRH; (NAcDNal¹, DCpa²,DPaI³,PicLys⁵,DOrn(ACyp)⁶,ILys⁸,DAla¹⁰)LHRH; N-acetyl-D-beta-Nal-D-Phe-D-Phe-Ser-Tyr-D-Lys(cyclo-pentyl)-Phe-Arg-Pro-D-Ala-NH₂; N-acetyl-D-ø-Nal-D-Phe-D-Phe-Ser-Tyr-D-Lys(cyclopentyl)-Phe-Lys(cyclopentyl)-Pro-D-Ala-NH₂; N-acetyl-D-beta-Nal-D-Phe-D-Phe-Ser-Tyr-D-Arg-Phe-(isopropyl)D-Lys-Pro-D-Ala-NH₂; N-acety1-D-beta-Nal-D-Phe-D-Phe-Ser-Tyr-D-Lys(benzyl)-Phe-Arg-Pro-D-Ala-NH₂; N-acetyl-D-beta-Nal-D-Phe-D-Phe-Ser-Tyr-D-Lys(Cl-benzyl)-Phe-Arg-Pro-D-Ala-NH₂; N-acetyl-D-beta-Nal-D-Phe-D-Phe-Ser-Tyr-D-Lys(heptyl)-Phe-Arg-Pro-D-Ala-NH₂; N-acetyl-D-beta-Nal-D-Phe-D-Phe-Ser-Tyr-D-Arg-Phe-Lys-(t-butylmethyl)-Pro-D-Ala-NH₂; N-acetyl-D-beta-Nal-D-Phe-D-Phe-Ser-Tyr-D-Arg-Phe-Lys-(4-methyl-benzyl)-Pro-D-Ala-NH₂; N-acetyl-D-beta-Nal-D-Phe-D-Phe-Ser-Tyr-D-Arg-Phe-Lys-(benzyl)-Pro-D-Ala-NH₂; N-acetyl-D-beta-Nal-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-p-NH₂-Phe-Phe-(isopropyl)Lys-Pro-D-Ala-NH₂; N-acetyl-D-beta-Nal-D-Phe-D-Phe-Ser-Tyr-D-Lys(heptyl)-Phe-Lys-(heptyl)-Pro-D-Ala-NH₂; N-acetyl-D-3-Nal-D-Phe-D-Phe-Ser-Tyr-D-Lys(1-butylpentyl)-Phe-Lys(1-butylpentyl)-Arg-Pro-D-Ala-NH₂,

The polypeptides of the present invention lack a functional H_(C) domain of a clostridial neurotoxin. Accordingly, said polypeptides are not able to bind rat synaptosomal membranes (via a clostridial H_(C) component) in binding assays as described in Shone et al. (1985) Eur. J. Biochem. 151, 75-82. In a preferred embodiment, the polypeptides preferably lack the last 50 C-terminal amino acids of a clostridial neurotoxin holotoxin. In another embodiment, the polypeptides preferably lack the last 100, preferably the last 150, more preferably the last 200, particularly preferably the last 250, and most preferably the last 300 C-terminal amino acid residues of a clostridial neurotoxin holotoxin. Alternatively, the Hc binding activity may be negated/reduced by mutagenesis—by way of example, referring to BoNT/A for convenience, modification of one or two amino acid residue mutations (W1266 to L and Y1267 to F) in the ganglioside binding pocket causes the H_(C) region to lose its receptor binding function. Analogous mutations may be made to non-serotype A clostridial peptide components, e.g. a construct based on botulinum B with mutations (W1262 to L and Y1263 to F) or botulinum E (W1224 to L and Y1225 to F). Other mutations to the active site achieve the same ablation of H_(C) receptor binding activity, e.g. Y1267S in botulinum type A toxin and the corresponding highly conserved residue in the other clostridial neurotoxins. Details of this and other mutations are described in Rummel et al (2004) (Molecular Microbiol. 51:631-634), which is hereby incorporated by reference thereto.

In another embodiment, the polypeptides of the present invention lack a functional H_(C) domain of a clostridial neurotoxin and also lack any functionally equivalent TM. Accordingly, said polypeptides lack the natural binding function of a clostridial neurotoxin and are not able to bind rat synaptosomal membranes (via a clostridial H_(C) component, or via any functionally equivalent TM) in binding assays as described in Shone et al. (1985) Eur. J. Biochem. 151, 75-82.

The H_(C) peptide of a native clostridial neurotoxin comprises approximately 400-440 amino acid residues, and consists of two functionally distinct domains of approximately 25 kDa each, namely the N-terminal region (commonly referred to as the H_(CN) peptide or domain) and the C-terminal region (commonly referred to as the H_(CC) peptide or domain). This fact is confirmed by the following publications, each of which is herein incorporated in its entirety by reference thereto: Umland T C (1997) Nat. Struct. Biol. 4: 788-792; Herreros J (2000) Biochem. J. 347: 199-204; Halpern J (1993) J. Biol. Chem. 268: 15, pp. 11188-11192; Rummel A (2007) PNAS 104: 359-364; Lacey DB (1998) Nat. Struct. Biol. 5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25: 90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7: 1751-1759; and Rummel A (2004) Mol. Microbiol. 51(3), 631-643. Moreover, it has been well documented that the C-terminal region (H_(CC)), which constitutes the C-terminal 160-200 amino acid residues, is responsible for binding of a clostridial neurotoxin to its natural cell receptors, namely to nerve terminals at the neuromuscular junction—this fact is also confirmed by the above publications. Thus, reference throughout this specification to a clostridial heavy-chain lacking a functional heavy chain H_(C) peptide (or domain) such that the heavy-chain is incapable of binding to cell surface receptors to which a native clostridial neurotoxin binds means that the clostridial heavy-chain simply lacks a functional H_(CC) peptide. In other words, the H_(CC) peptide region is either partially or wholly deleted, or otherwise modified (e.g. through conventional chemical or proteolytic treatment) to inactivate its native binding ability for nerve terminals at the neuromuscular junction.

Thus, in one embodiment, a clostridial H_(N) peptide of the present invention lacks part of a C-terminal peptide portion (H_(CC)) of a clostridial neurotoxin and thus lacks the H_(C) binding function of native clostridial neurotoxin. By way of example, in one embodiment, the C-terminally extended clostridial H_(N) peptide lacks the C-terminal 40 amino acid residues, or the C-terminal 60 amino acid residues, or the C-terminal 80 amino acid residues, or the C-terminal 100 amino acid residues, or the C-terminal 120 amino acid residues, or the C-terminal 140 amino acid residues, or the C-terminal 150 amino acid residues, or the C-terminal 160 amino acid residues of a clostridial neurotoxin heavy-chain. In another embodiment, the clostridial H_(N) peptide of the present invention lacks the entire C-terminal peptide portion (H_(CC)) of a clostridial neurotoxin and thus lacks the H_(C) binding function of native clostridial neurotoxin. By way of example, in one embodiment, the clostridial H_(N) peptide lacks the C-terminal 165 amino acid residues, or the C-terminal 170 amino acid residues, or the C-terminal 175 amino acid residues, or the C-terminal 180 amino acid residues, or the C-terminal 185 amino acid residues, or the C-terminal 190 amino acid residues, or the C-terminal 195 amino acid residues of a clostridial neurotoxin heavy-chain. By way of further example, the clostridial H_(N) peptide of the present invention lacks a clostridial H_(CC) reference sequence selected from the group consisting of:

-   -   Botulinum type A neurotoxin—amino acid residues (Y1111-L1296)     -   Botulinum type B neurotoxin—amino acid residues (Y1098-E1291)     -   Botulinum type C neurotoxin—amino acid residues (Y1112-E1291)     -   Botulinum type D neurotoxin—amino acid residues (Y1099-E1276)     -   Botulinum type E neurotoxin—amino acid residues (Y1086-K1252)     -   Botulinum type F neurotoxin—amino acid residues (Y1106-E1274)     -   Botulinum type G neurotoxin—amino acid residues (Y1106-E1297)     -   Tetanus neurotoxin—amino acid residues (Y1128-D1315).

The above-identified reference sequences should be considered a guide as slight variations may occur according to sub-serotypes.

The protease of the present invention embraces all non-cytotoxic proteases that are capable of cleaving one or more proteins of the exocytic fusion apparatus in eukaryotic cells.

The protease of the present invention is preferably a bacterial protease (or fragment thereof). More preferably the bacterial protease is selected from the genera Clostridium or Neisseria/Streptococcus (e.g. a clostridial L-chain, or a neisserial IgA protease preferably from N. gonorrhoeae or S. pneumoniae).

The present invention also embraces variant non-cytotoxic proteases (ie. variants of naturally-occurring protease molecules), so long as the variant proteases still demonstrate the requisite protease activity. By way of example, a variant may have at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95 or at least 98% amino acid sequence homology with a reference protease sequence. Thus, the term variant includes non-cytotic proteases having enhanced (or decreased) endopeptidase activity—particular mention here is made to the increased K_(cat)/K_(m) of BoNT/A mutants Q161A, E54A, and K165L see Ahmed, S. A. (2008) Protein J. DOI 10.1007/s10930-007-9118-8, which is incorporated by reference thereto. The term fragment, when used in relation to a protease, typically means a peptide having at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300 amino acid residues of the reference protease. As with the TM ‘fragment’ component (discussed above), protease ‘fragments’ of the present invention embrace fragments of variant proteases based on a reference sequence.

The protease of the present invention preferably demonstrates a serine or metalloprotease activity (e.g. endopeptidase activity). The protease is preferably specific for a SNARE protein (e.g. SNAP-25, synaptobrevin/VAMP, or syntaxin).

Particular mention is made to the protease domains of neurotoxins, for example the protease domains of bacterial neurotoxins. Thus, the present invention embraces the use of neurotoxin domains, which occur in nature, as well as recombinantly prepared versions of said naturally-occurring neurotoxins.

Exemplary neurotoxins are produced by clostridia, and the term clostridial neurotoxin embraces neurotoxins produced by C. tetani (TeNT), and by C. botulinum (BoNT) serotypes A-G, as well as the closely related BoNT-like neurotoxins produced by C. baratii and C. butyricum. The above-mentioned abbreviations are used throughout the present specification. For example, the nomenclature BoNT/A denotes the source of neurotoxin as BoNT (serotype A). Corresponding nomenclature applies to other BoNT serotypes.

BoNTs are the most potent toxins known, with median lethal dose (LD50) values for mice ranging from 0.5 to 5 ng/kg depending on the serotype. BoNTs are adsorbed in the gastrointestinal tract, and, after entering the general circulation, bind to the presynaptic membrane of cholinergic nerve terminals and prevent the release of their neurotransmitter acetylcholine. BoNT/B, BoNT/D, BoNT/F and BoNT/G cleave synaptobrevin/vesicle-associated membrane protein (VAMP); BoNT/C, BoNT/A and BoNT/E cleave the synaptosomal-associated protein of 25 kDa (SNAP-25); and BoNT/C cleaves syntaxin.

BoNTs share a common structure, being di-chain proteins of ˜150 kDa, consisting of a heavy chain (H-chain) of ˜100 kDa covalently joined by a single disulphide bond to a light chain (L-chain) of ˜50 kDa. The H-chain consists of two domains, each of ˜50 kDa. The C-terminal domain (H_(C)) is required for the high-affinity neuronal binding, whereas the N-terminal domain (H_(N)) is proposed to be involved in membrane translocation. The L-chain is a zinc-dependent metalloprotease responsible for the cleavage of the substrate SNARE protein.

The term L-chain fragment means a component of the L-chain of a neurotoxin, which fragment demonstrates a metalloprotease activity and is capable of proteolytically cleaving a vesicle and/or plasma membrane associated protein involved in cellular exocytosis.

Examples of suitable protease (reference) sequences include:

Botulinum type A neurotoxin amino acid residues (1-448) Botulinum type B neurotoxin amino acid residues (1-440) Botulinum type C neurotoxin amino acid residues (1-441) Botulinum type D neurotoxin amino acid residues (1-445) Botulinum type E neurotoxin amino acid residues (1-422) Botulinum type F neurotoxin amino acid residues (1-439) Botulinum type G neurotoxin amino acid residues (1-441) Tetanus neurotoxin amino acid residues (1-457) IgA protease amino acid residues (1-959)* *Pohlner, J. et al. (1987). Nature 325, pp. 458-462, which is hereby incorporated by reference thereto.

The above-identified reference sequence should be considered a guide as slight variations may occur according to sub-serotypes. By way of example, US 2007/0166332 (hereby incorporated by reference thereto) cites slightly different clostridial sequences:

Botulinum type A neurotoxin amino acid residues (M1-K448) Botulinum type B neurotoxin amino acid residues (M1-K441) Botulinum type C neurotoxin amino acid residues (M1-K449) Botulinum type D neurotoxin amino acid residues (M1-R445) Botulinum type E neurotoxin amino acid residues (M1-R422) Botulinum type F neurotoxin amino acid residues (M1-K439) Botulinum type G neurotoxin amino acid residues (M1-K446) Tetanus neurotoxin amino acid residues (M1-A457)

A variety of clostridial toxin fragments comprising the light chain can be useful in aspects of the present invention with the proviso that these light chain fragments can specifically target the core components of the neurotransmitter release apparatus and thus participate in executing the overall cellular mechanism whereby a clostridial toxin proteolytically cleaves a substrate. The light chains of clostridial toxins are approximately 420-460 amino acids in length and comprise an enzymatic domain. Research has shown that the entire length of a clostridial toxin light chain is not necessary for the enzymatic activity of the enzymatic domain. As a non-limiting example, the first eight amino acids of the BoNT/A light chain are not required for enzymatic activity. As another non-limiting example, the first eight amino acids of the TeNT light chain are not required for enzymatic activity. Likewise, the carboxyl-terminus of the light chain is not necessary for activity. As a non-limiting example, the last 32 amino acids of the BoNT/A light chain (residues 417-448) are not required for enzymatic activity. As another non-limiting example, the last 31 amino acids of the TeNT light chain (residues 427-457) are not required for enzymatic activity. Thus, aspects of this embodiment can include clostridial toxin light chains comprising an enzymatic domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, at least 425 amino acids and at least 450 amino acids. Other aspects of this embodiment can include clostridial toxin light chains comprising an enzymatic domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, at most 425 amino acids and at most 450 amino acids.

The polypeptides of the present invention, especially the protease component thereof, may be PEGylated—this may help to increase stability, for example duration of action of the protease component. PEGylation is particularly preferred when the protease comprises a BoNT/A, B or C₁ protease. PEGylation preferably includes the addition of PEG to the N-terminus of the protease component. By way of example, the N-terminus of a protease may be extended with one or more amino acid (e.g. cysteine) residues, which may be the same or different. One or more of said amino acid residues may have its own PEG molecule attached (e.g. covalently attached) thereto. An example of this technology is described in WO2007/104567, which is incorporated in its entirety by reference thereto.

A Translocation Domain is a molecule that enables translocation of a protease into a target cell such that a functional expression of protease activity occurs within the cytosol of the target cell. Whether any molecule (e.g. a protein or peptide) possesses the requisite translocation function of the present invention may be confirmed by any one of a number of conventional assays.

For example, Shone C. (1987) describes an in vitro assay employing liposomes, which are challenged with a test molecule. Presence of the requisite translocation function is confirmed by release from the liposomes of K⁺ and/or labelled NAD, which may be readily monitored [see Shone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180].

A further example is provided by Blaustein R. (1987), which describes a simple in vitro assay employing planar phospholipid bilayer membranes. The membranes are challenged with a test molecule and the requisite translocation function is confirmed by an increase in conductance across said membranes [see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp. 115-120].

Additional methodology to enable assessment of membrane fusion and thus identification of Translocation Domains suitable for use in the present invention are provided by Methods in Enzymology Vol 220 and 221, Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

The present invention also embraces variant translocation domains, so long as the variant domains still demonstrate the requisite translocation activity. By way of example, a variant may have at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% or at least 98% amino acid sequence homology with a reference translocation domain. The term fragment, when used in relation to a translocation domain, means a peptide having at least 20, preferably at least 40, more preferably at least 80, and most preferably at least 100 amino acid residues of the reference translocation domain. In the case of a clostridial translocation domain, the fragment preferably has at least 100, preferably at least 150, more preferably at least 200, and most preferably at least 250 amino acid residues of the reference translocation domain (eg. H_(N) domain). As with the TM ‘fragment’ component (discussed above), translocation ‘fragments’ of the present invention embrace fragments of variant translocation domains based on the reference sequences.

The Translocation Domain is preferably capable of formation of ion-permeable pores in lipid membranes under conditions of low pH. Preferably it has been found to use only those portions of the protein molecule capable of pore-formation within the endosomal membrane.

The Translocation Domain may be obtained from a microbial protein source, in particular from a bacterial or viral protein source. Hence, in one embodiment, the Translocation Domain is a translocating domain of an enzyme, such as a bacterial toxin or viral protein.

It is well documented that certain domains of bacterial toxin molecules are capable of forming such pores. It is also known that certain translocation domains of virally expressed membrane fusion proteins are capable of forming such pores. Such domains may be employed in the present invention.

The Translocation Domain may be of a clostridial origin, such as the H_(N) domain (or a functional component thereof). H_(N) means a portion or fragment of the H-chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H-chain, or the domain corresponding to that fragment in the intact H-chain. The H-chain lacks the natural binding function of the H_(C) component of the H-chain. In this regard, the H_(C) function may be removed by deletion of the H_(C) amino acid sequence (either at the DNA synthesis level, or at the post-synthesis level by nuclease or protease treatment). Alternatively, the H_(C) function may be inactivated by chemical or biological treatment. Thus, the H-chain is incapable of binding to the Binding Site on a target cell to which native clostridial neurotoxin (i.e. holotoxin) binds.

Examples of suitable (reference) Translocation Domains include:

Botulinum type A neurotoxin amino acid residues (449-871) Botulinum type B neurotoxin amino acid residues (441-858) Botulinum type C neurotoxin amino acid residues (442-866) Botulinum type D neurotoxin amino acid residues (446-862) Botulinum type E neurotoxin amino acid residues (423-845) Botulinum type F neurotoxin amino acid residues (440-864) Botulinum type G neurotoxin amino acid residues (442-863) Tetanus neurotoxin amino acid residues (458-879)

The above-identified reference sequence should be considered a guide as slight variations may occur according to sub-serotypes. By way of example, US 2007/0166332 (hereby incorporated by reference thereto) cites slightly different clostridial sequences:

Botulinum type A neurotoxin amino acid residues (A449-K871) Botulinum type B neurotoxin amino acid residues (A442-S858) Botulinum type C neurotoxin amino acid residues (T450-N866) Botulinum type D neurotoxin amino acid residues (D446-N862) Botulinum type E neurotoxin amino acid residues (K423-K845) Botulinum type F neurotoxin amino acid residues (A440-K864) Botulinum type G neurotoxin amino acid residues (S447-S863) Tetanus neurotoxin amino acid residues (S458-V879)

In the context of the present invention, a variety of Clostridial toxin H_(N) regions comprising a translocation domain can be useful in aspects of the present invention with the proviso that these active fragments can facilitate the release of a non-cytotoxic protease (e.g. a clostridial L-chain) from intracellular vesicles into the cytoplasm of the target cell and thus participate in executing the overall cellular mechanism whereby a clostridial toxin proteolytically cleaves a substrate. The H_(N) regions from the heavy chains of Clostridial toxins are approximately 410-430 amino acids in length and comprise a translocation domain. Research has shown that the entire length of a H_(N) region from a Clostridial toxin heavy chain is not necessary for the translocating activity of the translocation domain. Thus, aspects of this embodiment can include clostridial toxin H_(N) regions comprising a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids and at least 425 amino acids. Other aspects of this embodiment can include clostridial toxin H_(N) regions comprising translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids and at most 425 amino acids.

For further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, we refer to Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

The term H_(N) embraces naturally-occurring neurotoxin H_(N) portions, and modified H_(N) portions having amino acid sequences that do not occur in nature and/or synthetic amino acid residues, so long as the modified H_(N) portions still demonstrate the above-mentioned translocation function.

Alternatively, the Translocation Domain may be of a non-clostridial origin. Examples of non-clostridial (reference) Translocation Domain origins include, but not be restricted to, the translocation domain of diphtheria toxin [O=Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp. 25-51], the translocation domain of Pseudomonas exotoxin type A [Prior et al. Biochemistry (1992) 31, 3555-3559], the translocation domains of anthrax toxin [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442], a variety of fusogenic or hydrophobic peptides of translocating function [Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al (1992) PNAS, 89, pp. 7934-7938], and amphiphilic peptides [Murata et al (1992) Biochem., 31, pp. 1986-1992]. The Translocation Domain may mirror the Translocation Domain present in a naturally-occurring protein, or may include amino acid variations so long as the variations do not destroy the translocating ability of the Translocation Domain.

Particular examples of viral (reference) Translocation Domains suitable for use in the present invention include certain translocating domains of virally expressed membrane fusion proteins. For example, Wagner et al. (1992) and Murata et al. (1992) describe the translocation (i.e. membrane fusion and vesiculation) function of a number of fusogenic and amphiphilic peptides derived from the N-terminal region of influenza virus haemagglutinin. Other virally expressed membrane fusion proteins known to have the desired translocating activity are a translocating domain of a fusogenic peptide of Semliki Forest Virus (SFV), a translocating domain of vesicular stomatitis virus (VSV) glycoprotein G, a translocating domain of SER virus F protein and a translocating domain of Foamy virus envelope glycoprotein. Virally encoded Aspike proteins have particular application in the context of the present invention, for example, the E1 protein of SFV and the G protein of the G protein of VSV.

Use of the (reference) Translocation Domains listed in Table (below) includes use of sequence variants thereof. A variant may comprise one or more conservative nucleic acid substitutions and/or nucleic acid deletions or insertions, with the proviso that the variant possesses the requisite translocating function. A variant may also comprise one or more amino acid substitutions and/or amino acid deletions or insertions, so long as the variant possesses the requisite translocating function.

Translocation Amino acid Domain source residues References Diphtheria 194-380 Silverman et al., 1994, J. toxin Biol. Chem. 269, 22524- 22532 London E., 1992, Biochem. Biophys. Acta., 1113, 25-51 Domain II of 405-613 Prior et al., 1992, pseudomonas Biochemistry 31, 3555-3559 exotoxin Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538 Influenza virus GLFGAIAGFIENGWE Plank et al., 1994, J. Biol. haemagglutinin GMIDGWYG (SEQ ID Chem. 269, 12918-12924 NO: 110), and Wagner et al., 1992, PNAS, Variants thereof 89, 7934-7938 Murata et al., 1992, Biochemistry 31, 1986-1992 Semliki Forest Translocation domain Kielian et al., 1996, J Cell virus fusogenic Biol. 134(4), 863-872 protein Vesicular 118-139 Yao et al., 2003, Virology Stomatitis virus 310(2), 319-332 glycoprotein G SER virus F Translocation domain Seth et al., 2003, J Virol protein 77(11) 6520-6527 Foamy virus Translocation domain Picard-Maureau et al., 2003, envelope J Virol. 77(8), 4722-4730 glycoprotein

The polypeptides of the present invention may further comprise a translocation facilitating domain. Said domain facilitates delivery of the non-cytotoxic protease into the cytosol of the target cell and are described, for example, in WO 08/008803 and WO 08/008805, each of which is herein incorporated by reference thereto.

By way of example, suitable translocation facilitating domains include an enveloped virus fusogenic peptide domain, for example, suitable fusogenic peptide domains include influenzavirus fusogenic peptide domain (eg. influenza A virus fusogenic peptide domain of 23 amino acids), alphavirus fusogenic peptide domain (eg. Semliki Forest virus fusogenic peptide domain of 26 amino acids), vesiculovirus fusogenic peptide domain (eg. vesicular stomatitis virus fusogenic peptide domain of 21 amino acids), respirovirus fusogenic peptide domain (eg. Sendai virus fusogenic peptide domain of 25 amino acids), morbiliivirus fusogenic peptide domain (eg. Canine distemper virus fusogenic peptide domain of 25 amino acids), avulavirus fusogenic peptide domain (eg. Newcastle disease virus fusogenic peptide domain of 25 amino acids), henipavirus fusogenic peptide domain (eg. Hendra virus fusogenic peptide domain of 25 amino acids), metapneumovirus fusogenic peptide domain (eg. Human metapneumovirus fusogenic peptide domain of 25 amino acids) or spumavirus fusogenic peptide domain such as simian foamy virus fusogenic peptide domain; or fragments or variants thereof.

By way of further example, a translocation facilitating domain may comprise a Clostridial toxin H_(CN) domain or a fragment or variant thereof. In more detail, a Clostridial toxin H_(CN) translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids. In this regard, a Clostridial toxin H_(CN) translocation facilitating domain preferably has a length of at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific (reference) examples include:

Botulinum type A neurotoxin amino acid residues (872-1110) Botulinum type B neurotoxin amino acid residues (859-1097) Botulinum type C neurotoxin amino acid residues (867-1111) Botulinum type D neurotoxin amino acid residues (863-1098) Botulinum type E neurotoxin amino acid residues (846-1085) Botulinum type F neurotoxin amino acid residues (865-1105) Botulinum type G neurotoxin amino acid residues (864-1105) Tetanus neurotoxin amino acid residues (880-1127)

The above sequence positions may vary a little according to serotype/subtype, and further examples of suitable (reference) Clostridial toxin H_(CN) domains include:

Botulinum type A neurotoxin amino acid residues (874-1110) Botulinum type B neurotoxin amino acid residues (861-1097) Botulinum type C neurotoxin amino acid residues (869-1111) Botulinum type D neurotoxin amino acid residues (865-1098) Botulinum type E neurotoxin amino acid residues (848-1085) Botulinum type F neurotoxin amino acid residues (867-1105) Botulinum type G neurotoxin amino acid residues (866-1105) Tetanus neurotoxin amino acid residues (882-1127)

Any of the above-described facilitating domains may be combined with any of the previously described translocation domain peptides that are suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain may be combined with non-clostridial translocation domain peptide or with clostridial translocation domain peptide. Alternatively, a Clostridial toxin H_(CN) translocation facilitating domain may be combined with a non-clostridal translocation domain peptide. Alternatively, a Clostridial toxin H_(CN) facilitating domain may be combined or with a clostridial translocation domain peptide, examples of which include:

Botulinum type A neurotoxin amino acid residues (449-1110) Botulinum type B neurotoxin amino acid residues (442-1097) Botulinum type C neurotoxin amino acid residues (450-1111) Botulinum type D neurotoxin amino acid residues (446-1098) Botulinum type E neurotoxin amino acid residues (423-1085) Botulinum type F neurotoxin amino acid residues (440-1105) Botulinum type G neurotoxin amino acid residues (447-1105) Tetanus neurotoxin amino acid residues (458-1127)

Sequence Homology:

Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M—A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).

Alignment scores for determining sequence identity A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0 −3 −1 4

The percent identity is then calculated as:

$\frac{{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {identical}\mspace{14mu} {matches}}{\begin{matrix} \left\lbrack {{length}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {longer}\mspace{14mu} {sequence}\mspace{14mu} {plus}\mspace{14mu} {the}} \right. \\ {{number}\mspace{14mu} {of}\mspace{14mu} {gaps}\mspace{14mu} {introduced}\mspace{14mu} {into}\mspace{14mu} {the}\mspace{14mu} {longer}} \\ \left. {{sequence}\mspace{14mu} {in}\mspace{14mu} {order}\mspace{14mu} {to}\mspace{14mu} {align}\mspace{14mu} {the}\mspace{14mu} {two}\mspace{14mu} {sequences}} \right\rbrack \end{matrix}} \times 100$

Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

Conservative Amino Acid Substitutions

Basic: arginine

-   -   lysine     -   histidine         Acidic: glutamic acid     -   aspartic acid         Polar: glutamine     -   asparagine         Hydrophobic: leucine     -   isoleucine     -   valine         Aromatic: phenylalanine     -   tryptophan     -   tyrosine         Small: glycine     -   alanine     -   serine     -   threonine     -   methionine

In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and α-methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for clostridial polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.

Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

There now follows a brief description of the Figures, which illustrate aspects and/or embodiments of the present invention.

FIG. 1—Purification of LH_(N)/D-CT-CST29 fusion protein

Using the methodology outlined in Example 3, a LH_(N)/D-CT-CST29 fusion protein was purified from E. coli BL21 (DE3) cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 200 mM imidazole, treated with enterokinase to activate the fusion protein and then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE. Lane 1: First nickel chelating Sepharose column eluant, Lane 2: Second nickel chelating Sepharose column eluant under non-reducing conditions, Lane 3: Second nickel chelating Sepharose column eluant under reducing conditions, Lane 4: Molecular mass markers (kDa).

FIG. 2—Purification of LH_(N)/A-CT-SST14 fusion protein

Using the methodology outlined in Example 3, an LH_(N)/A-CT-SST14 fusion protein was purified from E. coli BL21 (DE3) cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 200 mM imidazole, treated with Factor Xa to activate the fusion protein and then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE. Lane 1: First nickel chelating Sepharose column eluant, Lane 2: Molecular mass markers (kDa), Lanes 3-4: Second nickel chelating Sepharose column eluant under non-reducing conditions, Lanes 5-6: Second nickel chelating Sepharose column eluant under reducing conditions.

FIGS. 3-47—Box Plots showing up-regulation of SNARE protein mRNA in cancer cells

Statistical analysis of the differences in SNARE expression between normal and primary cancer tissues was completed through use of Oncomine algorithms (Compendia Bioscience, Ann Arbor, Mich., USA) and gene microarray analysis tool. SNARE mRNA expression was compared to the median expression of all other genes in the respective study, for which a Normalised expression value was generated. Only studies with analysis results with P<0.05 are illustrated. In more detail, FIGS. 3-10 illustrate SNAP-25 expression profiles in different cancer versus non-cancer cells. FIGS. 11-16 illustrate syntaxin-1 expression profiles in different cancer versus non-cancer cells. FIGS. 17-20 illustrate syntaxin-2 expression profiles in different cancer versus non-cancer cells. FIGS. 21-24 illustrate syntaxin-3 expression profiles in different cancer versus non-cancer cells. FIGS. 25-32 illustrate VAMP-1 expression profiles in different cancer versus non-cancer cells. FIGS. 33-38 illustrate VAMP-2 expression profiles in different cancer versus non-cancer cells. FIGS. 39-45 illustrate VAMP-3 expression profiles in different cancer versus non-cancer cells. In all cases, the data illustrate up-regulation of SNARE proteins in cancer cells. FIG. 46 illustrates a statistically significant difference in syntaxin-1mRNA expression level between patients with recurrent invasive ductal breast cancer at 5 years post diagnosis and those patients without recurrence. FIG. 47 illustrates a statistically significant difference in syntaxin-1mRNA expression level in breast cancer patients in whom a metastatic event has occurred at 5 years post diagnosis versus those without metastases. Data is presented in box-plot form (see FIG. 3 for explanation) and/or as a histogram, where each bar represents the normalised gene expression level in each patient tissue sample.

FIG. 48—Profiling cell surface receptor and SNARE protein expression in Renal Cell carcinoma lines by Western blot analysis.

Protein extracts from eight human renal cell carcinoma (RCC) lines grown in vitro were prepared in standard Laemmli sample buffer (lanes 3-10). Protein extracts prepared from primary cultures of rat spinal cord neurons and dorsal root ganglia were included as controls. Proteins were separated on 10% SDS-polyacrylamide gels and electrophoretically transferred to a nitrocellulose membrane. After blocking of the membrane, primary antisera were used to probe for each specific protein and detection was enabled by a peroxidise-conjugated anti-species IgG. The receptor proteins detected were ErbB receptor (epidermal growth factor receptor, EGF) and growth hormone releasing hormone receptor (GHRHR). The SNARE proteins tested were SNAP-25, syntaxin-2 and syntaxin-3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a protein loading control.

FIG. 49—Detection of SNAP-25 protein cleavage in a Renal Cell carcinoma line by Western blot analysis after 24 hr treatment with an EGF-LHA fusion.

FIG. 49 shows the effect of 24 hour treatment of 786-0 human RCC cells with a number of EGF-liganded fusion protein molecules. In more detail, EGF-LHA, at doses between 1 and 100 nM, generated cleaved SNAP-25 species whereas control molecules, specifically EGF-LHB (non-SNAP-25 targeting), a catalytically inactive form of EGF-LHA, EGF-LH-DE-A (referred to as ‘EGF-0’ from hereon in) and the ‘free’ EGF ligand, did not. Indicated concentrations are in nM.

FIG. 50—Inhibition of in vitro proliferation of a Renal Cell carcinoma line by an EGF-LHA fusion.

786-0 cells seeded into a cell culture vessel were counted in a pre-define region at 24 and 48 hours after treatment with 25 nM of EGF-liganded fusion proteins.

FIG. 51—Inhibition of FGF-2 secretion from a Renal Cell carcinoma line by an EGF-LHA fusion.

Culture media from 786-0 cells treated for 24 hours with EGF-liganded fusion proteins were analysed for Fibroblast Growth Factor-2 by standard methods. In more detail, EGF-LHA, at doses between 1 and 50 nM, demonstrated a dose-dependent decrease in FGF-2 levels present in the culture medium whereas control molecules, specifically the catalytically inactive form of EGF-LHA, EGF-0, and ‘free’ EGF ligand, did not. Indicated concentrations are in nM.

FIG. 52—Inhibition of in vitro proliferation of the A498 Renal Cell carcinoma line by an EGF-LHA fusion.

FIG. 52 shows the effect of an EGF-liganded fusion protein (EGFv3-LHA, 300 nM) on cellular proliferation of A498 cell line after 48 hours. Cells were stained using the tetrazolium salt WST as per standard protocols. The unliganded molecule LHA was included as a control.

FIG. 53—Inhibition of in vitro proliferation of the ACHN Renal Cell carcinoma line by an EGF-LHA fusion.

FIG. 53 shows the effect of an EGF-liganded fusion protein (EGFv3-LHA, at 300 nM) on cellular proliferation of ACHN cell line after 48 hours. Cells were stained using the tetrazolium salt WST as per standard protocols. The unliganded molecule LHA was included as a control.

FIG. 54—Inhibition of in vitro secretion of gastrin releasing peptide (GRP) from the small cell lung cancer cell line DMS-53 by an EGF-LHA fusion.

FIG. 54 shows the effect of an EGF-liganded fusion protein (EGFv3-LHA, at 150 nM) on GRP secretion from the DMS-53 cell line after treatment for 24 hr. Medium removed from the cells were analysed 48 hours after removal of fusion proteins. The catalytically inactive form of EGF-LHA, EGF-0, was included as a control, as was cycloheximide, a general protein synthesis inhibitor.

NOMENCLATURE

SST somatostatin TGF(A) transforming growth factor (alpha) GHRL ghrelin LEP leptin ET(A) endothelin-1 FLT vascular endothelial growth factor receptor CHRN(D) acetylcholine receptor (subunit delta) EPHA ephrin type-A receptor EFNA ephrin-A DLK1 delta-like protein 1 JAG jagged protein NRG neuregulin G-CSF granulocyte colony-stimulating factor AMF autocrine motility factor NMB neuromedin-B CCK cholecystokinin PDGF platelet-derived growth factor ADM adrenomedullin GDNF glial cell line-derived neurotrophic factor TrkA high affinity nerve growth factor FSH follicle-stimulating hormone CXCR C-X-C chemokine receptor CRLR calcitonin-receptor-like receptor PDF prostate differentiation factor MCP monocyte chemotactic protein KGF keratinocyte growth factor FLK1 vascular endothelial growth factor receptor 2 PDGFR platelet-derived growth factor receptor NOTCH neurogenic locus notch homolog protein DLL delta-like protein GHS growth hormone secretagogue c-MET hepatocyte growth factor c-kit mast/stem cell growth factor MGSA/GRO melanoma growth stimulatory activity/growth related gene BCGF B-cell growth factor GnRH gonadotropin-releasing hormone receptor Ang-2 angiopoietin-2 FGF fibroblast growth factor ErbB epidermal growth factor receptor family member VIPR vasoactive intestinal polypeptide receptor BRS bombesin receptor subtype GRP gastrin releasing peptide LIF leukaemia inhibitory factor GHRH growth hormone-releasing hormone IGF insulin-like growth factor CRHR-2 corticotropin releasing factor receptor-2 BB bombesin GH growth hormone IL interleukin VEGF vascular endothelial growth factor ACH acetylcholine CST cortistatin VPAC vasoactive intestinal peptide receptor GRPR gastrin releasing peptide receptor CTR calcitonin binding receptor EPO erythropoietin HB-EGF heparin-binding EGF-like growth factor HGF/SF hepatocyte growth factor/scatter factor SDF-1 stromal cell-derived factor 1 CXCL12 chemokine (C-X-C motif) ligand 12 (SDF-1) TNF tumour necrosis factor PGF placental growth factor Gran4 granulin-4 TIE2 angiopoietin receptor-2 LH luteinising hormone CCL CC chemokine ligand NT neurotrophin NTAK neuregulin-2 BAFF B-cell activating factor GM-CSF granulocyte-macrophage colony stimulating factor NGF nerve growth factor PACAP pituitary adenylate cyclase-activating peptide OB leptin NRP neuropilin receptor

Summary of Examples

Example 1 Preparation of a LHA backbone construct

Example 2 Construction of LHD-CT-CST29

Example 3 Expression and purification of a LHD-CT-CST29 fusion protein

Example 4 Construction of LHA-CP-EGF

Example 5 Expression and purification of a LHA-CP-EGF fusion protein

Example 6 Chemical conjugation of LH_(N)/A to GnRH TM

Example 7 Method for treating colorectal cancer

Example 8 Method for treating breast cancer

Example 9 Method for treating prostate cancer

Example 10 Method for treating lung carcinoid tumours

Example 11 Method for treating bladder cancer

Example 12 Method for treating small cell lung cancer

Example 13 Method for treating prostate cancer

Example 14 Method for treating cervical cancer

Example 15 Method for treating leukaemia

Example 16 Method for treating small cell lung cancer

Example 17 Method for treating pancreatic cancer

Example 18 Method for treating metastatic bone cancer

Example 19 Method for treating metastatic small cell lung cancer in the brain

Example 20 Method for treating bowel cancer

Example 21 Method for treating chronic lymphocytic leukaemia

Example 22 Method for treating liver cancer

Example 23 Method for treating Hodgkin's lymphoma

Example 24 Method for treating renal cancer

Example 25 Method for treating skin cancer

Example 26 Method for treating oropharyngeal cancer

Example 27 Method for treating myeloma cancer

Example 28 Method for treating soft tissue sarcoma cancer

Example 29 Method for treating gastric cancer

Example 30 Method for treating testicular cancer

Example 31 Method for treating uterine cancer

Example 32 Method for treating Karposi sarcoma

Example 33 Method for treating primary brain cancer

Example 34 Method for treating rectal cancer

Example 35 Assessment of proliferation changes, inhibition of cellular secretion and concomitant SNARE cleavage after treatment of in vitro cultured renal cancer cell lines with a polypeptide of the present invention.

Summary of SEQ ID NOs

Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue/codon is optional.

-   -   1. DNA sequence of LH_(N)/A     -   2. DNA sequence of LH_(N)/B     -   3. DNA sequence of LH_(N)/C     -   4. DNA sequence of LH_(N)/D     -   5. DNA sequence of the CT-CST29 linker     -   6. DNA sequence of the LHD-CT-CST29 fusion     -   7. Protein sequence of the LHD-CT-CST29 fusion     -   8. DNA sequence of the CP-EGF linker     -   9. DNA sequence of the LHA-CP-EGF fusion     -   10. Protein sequence of the LHA-CP-EGF fusion     -   11. Protein sequence of LH_(N)/A     -   12. Protein sequence of LH_(N)/B     -   13. Protein sequence of LH_(N)/C     -   14. Protein sequence of LH_(N)/D     -   15.Synthesised GnRH peptide     -   16. Protein sequence of the LHB-CT-SST28 fusion     -   17. Protein sequence of the LHA-CP-SST28 fusion     -   18. Protein sequence of the LHD-CT-EGF fusion     -   19. Protein sequence of the LHD-CT-VIP fusion     -   20. Protein sequence of the LHC-CT-IGF1 fusion     -   21. Protein sequence of the LHD-CT-IGF1 fusion     -   22. Protein sequence of the LHC-CT-VIP fusion     -   23. Protein sequence of the LHC-CT-GnRH fusion     -   24. Protein sequence of the LHD-CT-GnRH fusion     -   25. Protein sequence of the LHD-CT-GRP fusion     -   26. Protein sequence of the LHB-CT-GRP fusion     -   27. Protein sequence of the LHC-CT-LIF fusion     -   28. Protein sequence of the LHB-CP-LIF fusion     -   29. Protein sequence of the LHC-CT-FGF1 fusion     -   30. Protein sequence of the LHA-CP-FGF1 fusion     -   31. Protein sequence of the LHA-CT-FGF9 fusion     -   32. Protein sequence of the LHC-CP-FGF9 fusion     -   33. Protein sequence of the IgA-H_(N)tet-CT-SST14 fusion     -   34. Protein sequence of the IgA-H_(N)tet-CP-SST14 fusion     -   35. Protein sequence of the LHA-CT-SST14 fusion     -   36. Protein sequence of the LHA-CT-EGFv3 fusion     -   37. Protein sequence of the LHE-CT-IL6 fusion     -   38. Protein sequence of the LHB-CT-IL8 fusion     -   39. Protein sequence of the LHF-CP-GRAN4 fusion     -   40. Protein sequence of the LHD-CP-TGFa fusion     -   41. Protein sequence of the LHD-CP-TGFb fusion     -   42. Protein sequence of the LHB-CT-TNFa fusion     -   43. Protein sequence of the LHD-CT-SDF1 fusion     -   44. Protein sequence of the LHC-CT-VEGF fusion

EXAMPLES Example 1 Preparation of a LH_(N)/a Backbone Construct

The following procedure creates a clone for use as an expression backbone for multidomain protein expression. This example is based on preparation of a serotype A based clone (SEQ ID1), though the procedures and methods are equally applicable to all LH_(N) serotypes such as serotype B (SEQ ID2), serotype C (SEQ ID3) and serotype D (SEQ ID4) and other protease or translocation domains by using the appropriate published sequence for synthesis.

Preparation of Cloning and Expression Vectors

pCR 4 (Invitrogen) is the chosen standard cloning vector chosen due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation. The expression vector is based on the pET (Novagen) expression vector which has been modified to contain the multiple cloning site NdeI-BamHI-SalI-PstI-XbaI-HindIII for construct insertion, a fragment of the expression vector has been removed to create a non-mobilisable plasmid, a variety of different fusion tags have been inserted to increase purification options and an existing XbaI site in the vector backbone has been removed to simplify sub-cloning.

Preparation of LC/A

The DNA sequence is designed by back translation of the LC/A amino acid sequence (obtained from freely available database sources such as GenBank (accession number P10845) using one of a variety of reverse translation software tools (for example Backtranslation tool v2.0 (Entelechon)). BamHI/SalI recognition sequences are incorporated at the 5′ and 3′ ends respectively of the sequence maintaining the correct reading frame. The DNA sequence is screened (using software such as SeqBuilder, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed by the Backtranslation tool from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (GeneArt), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, Sep. 13, 2004). This optimised DNA sequence containing the LC/A open reading frame (ORF) is then commercially synthesized (for example by Entelechon, GeneArt or Sigma-Genosys) and is provided in the pCR 4 vector.

Preparation of H_(N)/a Insert

The DNA sequence is designed by back translation of the H_(N)/A amino acid sequence (obtained from freely available database sources such as GenBank (accession number P10845) using one of a variety of reverse translation software tools (for example Back translation tool v2.0 (Entelechon)). A PstI restriction sequence added to the N-terminus and XbaI-stop codon-HindIII to the C-terminus ensuring the correct reading frame in maintained. The DNA sequence is screened (using software such as SeqBuilder, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed by the Backtranslation tool from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (GeneArt), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, Sep. 13, 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, GeneArt or Sigma-Genosys) and is provided in the pCR 4 vector.

Preparation of the Interdomain (LC-H_(N) Linker)

The LC-H_(N) linker can be designed from first principle, using the existing sequence information for the linker as the template. For example, the serotype A linker (in this case defined as the inter-domain polypeptide region that exists between the cysteines of the disulphide bridge between LC and H_(N)) has the sequence VRGIIPFKTKSLDEGYNKALNDL (SEQ ID NO: 111). This sequence information is freely available from available database sources such as GenBank (accession number P10845). For generation of a specific protease cleavage site, the native recognition sequence for Factor Xa can be used in the modified sequence VDGIITSKTKSLIEGRNKALNLQ (SEQ ID NO: 112) or an enterokinase recognition sequence is inserted into the activation loop to generate the sequence VDGIITSKTKSDDDDKNKALNLQ SEQ ID NO: 113). Using one of a variety of reverse translation software tools (for example Backtranslation tool v2.0 (Entelechon), the DNA sequence encoding the linker region is determined. BamHI/SalI and PstI/XbaI/stop codon/HindIII restriction enzyme sequences are incorporated at either end, in the correct reading frames. The DNA sequence is screened (using software such as Seqbuilder, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed by the Backtranslation tool from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (GeneArt), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, Sep. 13, 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, GeneArt or Sigma-Genosys) and is provided in the pCR 4 vector.

Assembly and Confirmation of the Backbone Clone

Due to the small size, the activation linker must be transferred using a two step process. The pCR-4 linker vector is cleaved with BamHI+SalI combination restriction enzymes and the cleaved linker vector then serves as the recipient for BamHI+SalI restriction enzyme cleaved LC DNA. Once the LC encoding DNA is inserted upstream of the linker DNA, the entire LC-linker DNA fragment can then be isolated and transferred to the pET expression vector MCS. The LC-linker is cut out from the pCR 4 cloning vector using BamHI/PstI restriction enzymes digests. The pET expression vector is digested with the same enzymes but is also treated with antarctic phosphatase as an extra precaution to prevent re-circularisation. The LC-linker and the pET vector backbone are gel purified and the purified insert and vector backbone are ligated together using T4 DNA ligase. The product is transformed with TOP10 cells which are then screened for LC-linker using BamHI/PstI restriction digestion. The process is then repeated for the H_(N) insertion into the PstI/HindIII restriction sites of the pET-LC-linker construct.

Screening with restriction enzymes is sufficient to ensure the final backbone is correct as all components are already sequenced confirmed during synthesis. However, during the sub-cloning of some components into the backbone, where similar size fragments are being removed and inserted, sequencing of a small region to confirm correct insertion is required.

Example 2 Construction of LH_(N)/D-CT-CST29

The following procedure creates a clone for use as an expression construct for multidomain fusion expression where the targeting moiety (TM) is presented C-terminally to the translocation domain. This example is based on preparation of the LH_(N)/D-CT-CST29 fusion (SEQ ID6), though the procedures and methods are equally applicable to create other protease, translocation and TM fusions, where the TM of C-terminal to the translocation domain. In this example, a flanking 15 amino acid glycine-serine spacer is engineered into the interdomain sequence to ensure accessibility of the ligand to its receptor, but other spacers are applicable.

Preparation of Spacer-CST29 Insert

For presentation of a CST29 sequence at the C-terminus of the H_(N) domain, a DNA sequence is designed to flank the spacer and targeting moiety (TM) regions allowing incorporation into the backbone clone (SEQ ID4). The DNA sequence can be arranged as BamHI-SalI-PstI-XbaI-spacer-CST29-stop codon-HindIII (SEQ ID5). The DNA sequence can be designed using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)). Once the TM DNA is designed, the additional DNA required to encode the preferred spacer is created in silico. It is preferred to ensure the correct reading frame is maintained for the spacer, TM and restriction sequences and that the XbaI sequence is not preceded by the bases TC, which would result in DAM methylation. The DNA sequence is screened for restriction sequences incorporated and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (GeneArt), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, Sep. 13, 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, GeneArt or Sigma-Genosys) and is provided in the pCR 4 vector.

Assembly and Confirmation of the Backbone Clone

In order to create a LH_(N)/D-CT-CST29 construct (SEQ ID6) using the backbone construct (SEQ ID4) and the newly synthesised pCR 4-spacer-TM vector encoding the CST29 TM (SEQ ID5), a one or two step method can be used; typically the two step method is used when the TM DNA is less than 100 base pairs. Using the one step method the TM can be inserted directly into the backbone construct buy cutting the pCR 4-spacer-TM vector with XbaI and HindIII restriction enzymes and inserting the TM encoding DNA fragment into a similarly cut pET backbone construct. Using the two-step method the LH_(N) domain is excised from the backbone clone using restriction enzymes BamHI and XbaI and ligated into similarly digested pCR 4-spacer-TM vector. This creates an LH_(N)-spacer-TM ORF in pCR 4 that can be excised from the vector using restriction enzymes BamHI and HindIII for subsequent ligation into the similarly cleaved pET expression construct. The final construct contains the LC-linker-H_(N)-spacer-CST29 DNA (SEQ ID6) which will result in a fusion protein containing the sequence illustrated in SEQ ID7.

Example 3 Expression and Purification of a LH_(N)/D-CT-CST29 Fusion Protein

This example is based on preparation of an LH_(N)/D protein that incorporates a CST29 TM polypeptide at the carboxyl terminus of the H_(N) domain (SEQ ID7), where the pET expression vector ORF also encodes a histidine purification tag. These procedures and methods are equally applicable to fusion protein sequences shown in SEQ ID 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 31, 33, 35, 36, 37, 38, 42, 43 or 44. Where appropriate, the activation enzyme should be selected to be compatible with the protease activation site within each sequence.

Expression of LH_(N)/D-CT-CST29

Expression of the LH_(N)/D-CT-CST29 protein is achieved using the following protocol. Inoculate 100 ml of modified TB containing 0.2% glucosamine and 30 μg/ml kanamycin in a 250 ml flask with a single colony from the LH_(N)/D-CT-CST29 expression strain. Grow the culture at 37° C., 225 rpm for 16 hours. Inoculate 1 L of modified TB containing 0.2% glucosamine and 30 μg/ml kanamycin in a 2 L flask with 10 ml of overnight culture. Grow cultures at 37° C. until an approximate OD₆₀₀ nm of 0.5 is reached at which point reduce the temperature to 16° C. After 1 hour induce the cultures with 1 mM IPTG and grow at 16° C. for a further 16 hours.

Purification of LH_(N)/D-CT-CST29 Protein

Defrost falcon tube containing 35 ml 50 mM HEPES pH 7.2 200 mM NaCl and approximately 10 g of E. coli BL21 (DE3) cell paste. Homogenise the cell paste (20 psi) ensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4° C. for 30 minutes. Load the supernatant onto a 0.1 M NiSO₄ charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2 200 mM NaCl. Using a step gradient of 10, 40 and 100 mM imidazole, wash away the non-specific bound protein and elute the fusion protein with 200 mM imidazole. The eluted fusion protein is dialysed against 5 L of 50 mM HEPES pH 7.2 200 mM NaCl at 4° C. overnight and the OD₂₈₀ nm measured to establish the protein concentration. Add 3.2 μl enterokinase (New England Biolabs) per mg fusion protein and incubate static overnight at 25° C. Load onto a 0.1 M NiSO₄ charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2 200 mM NaCl. Wash column to baseline with 50 mM HEPES pH 7.2 200 mM NaCl. Using a step gradient of 10, 40 and 100 mM imidazole, wash away the non-specific bound protein and elute the fusion protein with 200 mM imidazole. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2 150 mM NaCl at 4° C. overnight and concentrate the fusion to about 2 mg/ml, aliquot sample and freeze at −20° C. Test purified protein using OD₂₈₀, BCA and purity analysis. FIGS. 1 and 2 demonstrate purification of fusion proteins following this method as analysed by SDS-PAGE.

Example 4 Construction of LH_(N)/A-CP-EGF

The following procedure creates a clone for use as an expression construct for multidomain fusion expression where the targeting moiety (TM) is presented centrally between the protease and translocation domain. This example is based on preparation of the LH_(N)/A-CP-EGF fusion (SEQ ID9), though the procedures and methods are equally applicable to create other protease, translocation and TM fusions, where the TM is N-terminal to the translocation domain. In this example, a flanking helical spacer is engineered into the interdomain sequence to ensure accessibility of the ligand to its receptor, but other spacers are applicable.

Preparation of Spacer-Human EGF Insert

The LC-H_(N) inter-domain polypeptide linker region exists between the cysteines of the disulphide bridge between LC and H_(N). For insertion of a protease cleavage site, spacer and a targeting moiety (TM) region into the activation loop, one of a variety of reverse translation software tools (for example Backtranslation tool v2.0 (Entelechon) are used to determine the DNA sequence encoding the linker region. For central presentation of a TM sequence at the N-terminus of the H_(N) domain, a DNA sequence is designed for the spacer and targeting moiety (TM) regions allowing incorporation into the backbone clone (SEQ ID1). The DNA sequence can be arranged as BamHI-SalI-spacer-protease activation site-EGF-spacer-PstI-XbaI-stop codon-HindIII (SEQ ID8). Once the TM DNA is designed, the additional DNA required to encode the preferred spacer is created in silico. It is preferred to ensure the correct reading frame is maintained for the spacer, TM and restriction sequences and that the XbaI sequence is not preceded by the bases TC, which would result in DAM methylation. The DNA sequence is screened for restriction sequence incorporated and any additional sites are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (GeneArt), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, Sep. 13, 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, GeneArt or Sigma-Genosys) and is provided in the pCR 4 vector.

Assembly and Confirmation of the Backbone Clone

In order to create a LC-spacer-activation site-EGF-spacer-H_(N) construct (SEQ ID9) using the backbone construct (SEQ ID1) and the newly synthesised pCR 4-spacer-activation site-TM-spacer vector encoding the EGF TM (SEQ ID8), a one or two step method can be used; typically the two step method is used when the TM DNA is less than 100 base pairs. Using the one step method the TM linker region can be inserted directly into the backbone construct buy cutting the pCR 4-spacer-activation site-TM-spacer vector with SalI and PstI restriction enzymes and inserting the TM encoding DNA fragment into a similarly cut pET backbone construct. Using the two-step method the LC domain is excised from the backbone clone using restriction enzymes BamHI and SalI and ligated into similarly digested pCR 4-spacer-activation site-TM-spacer vector. This creates a LC-spacer-activation site-TM-spacer ORF in pCR 4 that can be excised from the vector using restriction enzymes BamHI and PstI for subsequent ligation into similarly pET expression construct. The final construct contains the LC-spacer-activation site-EGF-spacer-H_(N) DNA (SEQ ID9) which will result in a fusion protein containing the sequence illustrated in SEQ ID10.

Example 5 Expression and Purification of a LH_(N)/A-CP-EGF Fusion Protein

This example is based on preparation of an LH_(N)/A protein that incorporates a EGF TM polypeptide into the interdomain linker region (SEQ ID10), where the pET expression vector ORF also encodes a histidine purification tag. These procedures and methods are equally applicable to the other fusion protein shown in SEQ ID 17, 28, 30, 32, 34, 39, 40 or 41. Where appropriate, the activation enzyme should be selected to be compatible with the protease activation site within each sequence.

Expression of LH_(N)/A-CP-EGF

Expression of the LH_(N)/A-CP-EGF protein is achieved using the following protocol. Inoculate 100 ml of modified TB containing 0.2% glucosamine and 30 μg/ml kanamycin in a 250 ml flask with a single colony from the LHA-CP-EGF expression strain. Grow the culture at 37° C., 225 rpm for 16 hours. Inoculate 1 L of modified TB containing 0.2% glucosamine and 30 μg/ml kanamycin in a 2 L flask with 10 ml of overnight culture. Grow cultures at 37° C. until an approximate OD₆₀₀ nm of 0.5 is reached at which point reduce the temperature to 16° C. After 1 hour induce the cultures with 1 mM IPTG and grow at 16° C. for a further 16 hours.

Purification of LH_(N)/A-CP-EGF Protein

Defrost falcon tube containing 35 ml 50 mM HEPES pH 7.2 200 mM NaCl and approximately 10 g of E. coli BL21 (DE3) cell paste. Homogenise the cell paste (20 psi) ensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4° C. for 30 minutes. Load the supernatant onto a 0.1 M NiSO4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2 200 mM NaCl. Using a step gradient of 10, 40 and 100 mM imidazole, wash away the non-specific bound protein and elute the fusion protein with 200 mM imidazole. The eluted fusion protein is dialysed against 5 L of 50 mM HEPES pH 7.2 200 mM NaCl at 4° C. overnight and the OD₂₈₀ nm measured to establish the protein concentration. Add 3.2 μl enterokinase (New England Biolabs) per mg fusion protein and incubate static overnight at 25° C. Load onto a 0.1 M NiSO4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2 200 mM NaCl. Wash column to baseline with 50 mM HEPES pH 7.2 200 mM NaCl. Using a step gradient of 10, 40 and 100 mM imidazole, wash away the non-specific bound protein and elute the fusion protein with 200 mM imidazole. Dialyse the eluted fusion protein against 5L of 50 mM HEPES pH 7.2 150 mM NaCl at 4° C. overnight and concentrate the fusion to about 2 mg/ml, aliquot sample and freeze at −20° C. Test purified protein using OD₂₈₀, BCA and purity analysis.

Example 6 Chemical Conjugation of LH_(N)/A to GnRH TM

The following procedure creates a chemically conjugated molecule containing the LH_(N)/A amino acid sequence (SEQ ID11), prepared from SEQ ID1 using the production method outlined in example 3, and a GnRH peptide which has been chemically synthesised (SEQ ID15). However, the procedures and methods are equally applicable for the conjugation of other peptides to other protease/translocation domain proteins such as those containing the amino acid sequences SEQ ID12, 13 and 14.

The LH_(N)/A protein was buffer exchanged from 50 mM Hepes 150 mM salt into PBSE (100 mM 14.2 g NA2HPO4, 100 mM 5.85 g NaCl, 1 mM EDTANa₂ pH 7.5 with 1M HCl) using the Bio-rad PD10 column. This was done by washing one column volume of PBSE through the PD10 column, the protein was then added to the column until no more drops exit the end of the PD10 column. 8 mls of PBSE was then added and 0.5 ml fractions are collected. The collected fractions are the measured using the A₂₈₀ reading and fractions containing protein are pooled. A concentration of 1.55 mg/ml of LH_(N)/A was obtained from the buffer exchange step and this was used to set up the following reactions:

LH_(N)/A 1.55 mg/ml 20 mM SPDP or Sulfo-LC-SPDP A 200 μl 0 B 200 μl 4 fold increase 0.62 μl C 200 μl 8 fold increase 1.24 μl

Sample were left to tumble at RT for 3 hours before being passed down another PD10 column to buffer exchange into PBSE and the protein containing fractions pooled. A final concentration of 25 mM DTT was then added to derivatised protein and then the samples left at room temperature for 10 minutes. A₂₈₀ and A₃₄₃ readings were then taken to work out the ratio of SPDP:LH_(N)/A interaction and the reaction which resulted in a derivatisation ration of between 1 and 3 was used for the peptide conjugation. The SPDP reagent binds to the primary amines of the LH_(N)/A via an N-hydroxysuccinimide (NHS) ester, leaving the sulphydryl-reactive portion to form a disulphide bond to the free SH group on the free cysteine on the synthesised peptide. In this case the peptide sequence is GnRH which has been synthesised with a cysteine contained within the peptide to allow conjugation whilst leaving the N-terminus and C-terminus of the GnRH to interact with its receptor (SEQ ID15). The SPDP-derivatised LH_(N)/A was mixed with a 4-fold excess of the GnRH ligand and the reaction was then left at RT for 90 minutes whilst tumbling. The excess GnRH was then removed using either a PD10 column leaving LH_(N)/A-GnRH conjugated molecule.

Example 7—Method for Treating Colorectal Cancer

A 62 year old man presents with a stage II colorectal cancer. To reduce and/or to prevent metastasis he receives a direct injection of a polypeptide of the present invention (eg. botulinum type A neurotoxin protease and translocation domain and a VIP peptide). Within 4 weeks a significant shrinkage of the tumour is observed without appearance of metastasis elsewhere. The treatment is optionally performed in combination with chemotherapy, and is repeated 2 months later and 4 weeks later no tumour is observable anymore with the usual detection tools (colonoscopy, CT scan, PET scan, etc.) and the level of carcinoembryonic antigen (CEA) returned to the normal.

Example 8—Method for Treating Breast Cancer

A 61 year old woman presents with a stage II breast cancer. To treat and/or prevent metastasis she receives an IV injection of a polypeptide of the present invention (eg. a botulinum type C neurotoxin protease, a botulinum type C neurotoxin translocation domain and a GnRH peptide), optionally in combination with chemotherapy. Within 4 weeks a significant shrinkage of the tumour is observed without appearance of metastasis elsewhere. The treatment is repeated 2 months later and 6 weeks later no tumour is observable anymore with the usual detection tools (MRI, ultrasound, breast-specific positron emission tomography, mammography, Scintigraphy, etc).

Example 9—Method for Treating Prostate Cancer

A 77 year old man presents with a stage II prostate cancer. To treatment and/or to prevent metastasis he receives a intravenous injection of a polypeptide of the present invention (eg. a botulinum type C neurotoxin protease, a botulinum type C neurotoxin translocation domain and an IGF-1 peptide), optionally in combination with hormone therapy. Within 4 weeks a significant shrinkage of the tumour is observed without appearance of metastasis elsewhere. The treatment is repeated 2 months later and 8 weeks later no tumour is observable anymore with the usual detection tools (X-ray, ProstaScint scan, MRI, transrectal ultrasonography, CT scan, etc.) and the levels of PSA came back to normal.

Example 10—Method for Treating Lung Carcinoid Tumours

A 66 year old woman presents with lung carcinoid tumours. To treat and/or to prevent metastasis she receives an IV injection of a polypeptide of the present invention (eg. a botulinum type A neurotoxin protease, a botulinum type A neurotoxin translocation domain and a bFGF-1 peptide), optionally in combination with chemotherapy. Within 4 weeks a significant decrease in the size of the tumour is observed without appearance of metastasis elsewhere. The treatment is repeated 1 month later and 4 weeks later no tumour is observable anymore with the usual detection tools (X-rays, CT scan, bronchoscopy, etc.) or using the usual blood tests recommended for this cancer.

Example 11—Method for Treating Bladder Cancer

A 56 year old man presents with a stage II bladder cancer. To treat and/or to prevent metastasis he receives a direct injection of a polypeptide of the present invention (eg. an IgA protease, a tetanus neurotoxin translocation domain and an IGF-1 peptide), optionally in combination with chemotherapy. Within 2 weeks a significant shrinkage of the tumour is observed without appearance of metastasis elsewhere. The treatment is repeated 2 months later and 4 weeks later no tumour is observable anymore with the usual detection tools (colonoscopy, CT scan, PET scan, etc.).

Example 12—Method for Treating Small Cell Lung Cancer

A 79 year old man is diagnosed with a stage I small cell lung cancer. To treat and/or to prevent metastasis he receives a injection of a polypeptide of the present invention (eg. a botulinum type A neurotoxin protease, a botulinum type A neurotoxin translocation domain and a neuregulin ERBB3 peptide), optionally in combination with chemotherapy. Within 3 weeks a significant decrease in size of the tumour is observed without appearance of metastasis elsewhere. The treatment is repeated 2 months later and 5 weeks later no tumour is observable anymore with the usual detection tools (X-rays, CT scan, MRI, PET scanning, Radionuclide imaging, bronchoscopy, etc.) or using the usual blood tests recommended for this cancer.

Example 13—Method for Treating Prostate Cancer

A 72 year old man is diagnosed with a stage II prostate cancer. To treat and/or to prevent metastasis he receives a intravenous injection of a polypeptide of the present invention (eg. a botulinum type D neurotoxin protease, a botulinum type C neurotoxin translocation domain, a GS20 linker, and an IGF-1 peptide), optionally in combination with androgen deprivative treatment. Within 10 days a significant shrinkage of the tumour is observed without appearance of metastasis elsewhere. The treatment is repeated 2 months later and 5 weeks later no tumour is observable anymore with the usual detection tools (X-ray, ProstaScint scan, MRI, transrectal ultrasonography, CT scan, etc.) and the levels of PSA came back to normal.

Example 14—Method for Treating Cervical Cancer

A 60 year old woman diagnosed with cervical cancer at a limited stage is treated with surgery. To improve the effects of the treatment and to prevent metastasis she receives an intravenous injection of a polypeptide of the present invention (eg. botulinum type D neurotoxin protease, a botulinum type D neurotoxin translocation domain, a GS20 linker, and a somatostatin-14 peptide). Within 6 weeks no re-appearance of the tumour is observed. The treatment is repeated 3 months later and 8 weeks later no tumour is observable anymore with the usual detection tools (X-rays, CT scan, MRI, PET scanning, Radionuclide imaging, bronchoscopy, etc.) or using the usual blood tests recommended for this cancer.

Example 15—Method for Treating Leukaemia

A 42 year old man is diagnosed with a stage II Hairy cell leukaemia cancer. To treat and/or to prevent metastasis he receives an intravenous injection of a polypeptide of the present invention (eg. a botulinum type D neurotoxin protease, a botulinum type D neurotoxin translocation domain, a GS20 linker, and a FGF-1 peptide), optionally in combination with chemotherapy. Within 10 days significant reduction in tumour burden is observed without appearance of metastasis. The treatment is repeated 1 month later and 3 weeks later no tumour is observable anymore with the usual detection tools (MRI, complete blood count, etc).

Example 16—Method for Treating Small Cell Lung Cancer

A 56 year old man is diagnosed with a small cell lung cancer at an extensive stage. To treat and/or to prevent metastasis elsewhere he receives an intravenous injection of a polypeptide of the present invention (eg. a botulinum type D neurotoxin protease, a botulinum type C neurotoxin translocation domain and an EGF peptide), optionally in combination with chemotherapy and radiation therapy. Within 3 weeks a significant shrinkage of the tumour and a diminution in size of the metastasis is observed without appearance of new metastasis elsewhere. The treatment is repeated twice after 2 months and 5 months. The patients died 11 months later, 6 months later than expected with this type of treatment and this stage of the disease.

Example 17—Method for Treating Pancreatic Cancer

A 48 year old woman is diagnosed with pancreatic cancer at an advanced stage. She receives an intravenous injection of a polypeptide of the present invention (eg. a botulinum type D neurotoxin protease, a botulinum type C neurotoxin translocation domain and an EGF peptide), optionally in combination with the chemotherapeutic agent, Gemcitabine. Within 3 weeks a significant reduction in primary tumour growth and a diminution in size of the metastases are observed without appearance of new metastasis elsewhere. The treatment is repeated twice after 2 months and 5 months. The patient died 12 months later, 6 months later than expected with this type of treatment and this stage of the disease.

Example 18—Method for Treating Metastatic Bone Cancer

A 71 year old man is diagnosed with a stage IV prostate cancer. To treat metastatic growth in his bone he receives an intravenous injection of a polypeptide of the present invention (eg. a botulinum type D neurotoxin protease, a botulinum type D neurotoxin translocation domain and a TGF-beta peptide), optionally in combination with external beam radiation plus hormone therapy. Within 4 weeks a significant shrinkage of the tumour at the metastatic sites is observed without appearance of metastasis elsewhere. The treatment is repeated 2 and 4 months later. After 6 months no detectable increase in tumour burden is observable anymore with the usual detection tools (X-ray, ProstaScint scan, MRI, CT scan, etc.).

Example 19—Method for Treating Metastatic Small Cell Lung Cancer in the Brain

A 65 year old man diagnosed with a small cell lung cancer at an advanced stage also presents with multiple brain metastases. To treat, a polypeptide of the present invention (eg. a botulinum type A neurotoxin protease, a botulinum type A neurotoxin translocation domain, and a SDF-1 peptide) is delivered into his brain by convection enhanced delivery, optionally in combination with chemotherapy and whole brain radiation. Within 4 weeks significant shrinkage of the metastatic tumour sites is observed without appearance of metastasis elsewhere. The treatment is repeated at 2 months and 8 weeks later no metastatic tumour is observable anymore with the usual detection tools (X-rays, CT scan, MRI, PET scanning, Radionuclide imaging, etc.) or using the usual blood tests recommended for this cancer.

Example 20—Method for Treating Bowel Cancer

A 76 year old man is diagnosed with a stage II small bowel cancer. To treatment and/or to prevent metastasis he receives a direct injection of a polypeptide of the present invention (eg. a botulinum type A neurotoxin protease and translocation domain and an EGF peptide), optionally in combination with chemotherapy. Within 4 weeks a significant shrinkage of the tumour is observed without appearance of metastasis elsewhere and surgery is then realized to remove the tumour.

Example 21—Method for Treating Chronic Lymphocytic Leukaemia

A 54 year old man is diagnosed with relapsed chronic lymphocytic leukaemia. To treat, he receives an intravenous injection of a polypeptide of the present invention (eg. a botulinum type B neurotoxin protease, a botulinum type B neurotoxin translocation domain and an LIF-1 peptide), optionally in combination with chemotherapy. Within 2 weeks a significant reduction in tumour cell count in the patient's blood is observed which remains stable for an extended period of time as determined by microscopic examination and flow cytometric analysis of the patient's blood

Example 22—Method for Treating Liver Cancer

A 55 year old woman is diagnosed with a stage IV hepatic cancer. To treat, she receives an intravenous injection of a polypeptide of the present invention (eg. a botulinum type D neurotoxin protease, a botulinum type D neurotoxin translocation domain and a TGF-alpha peptide), optionally in combination with chemotherapy. Within 2 weeks a significant shrinkage of the tumour is observed on PET-CT imaging studies along with a reduction of alpha-fetoprotein in the blood, which was elevated prior to treatment.

Example 23—Method for Treating Hodgkin's Lymphoma

A 24 year old man is diagnosed with stage IVA Hodgkin's lymphoma. To treat, he receives repeated intravenous injections of a polypeptide of the present invention (eg. a botulinum type B neurotoxin protease, a botulinum type B neurotoxin translocation domain and an VEGF peptide), optionally in combination with chemotherapy. Within 2 weeks a significant reduction in tumour volume is observed and tumour blood flow using the usual detection tools (MRI, CT scans) and leads to complete remission within 2 months.

Example 24—Method for Treating Renal Cancer

A 54 year old man is diagnosed with advanced renal cell carcinoma. To treat, he receives an intravenous administration of a polypeptide of the present invention (eg. a botulinum type A neurotoxin protease, a botulinum type C neurotoxin translocation domain and a VEGF peptide), optionally in combination with the anti-angiogenic therapeutic, Sunitinib, a small molecule tyrosine kinase inhibitor (TKI). Within 14 days a significant shrinkage of the tumour over and above that expected with TKI alone is observed using the usual detection tools (CT, MRI scans, blood tests).

Example 25—Method for Treating Skin Cancer

A 31 year old woman is diagnosed with a facial melanoma. To treat, she receives direct administration of a polypeptide of the present invention (eg. a botulinum type D neurotoxin protease, a botulinum type C neurotoxin translocation domain, a GS20 linker, and a VEGF peptide), optionally in combination with chemotherapy and immunotherapy. Within 21 days a significant shrinkage of the tumour over and above that expected with chemo- and immunotherapy alone is observed, allowing surgical resection with clear but much reduced margins.

Example 26—Method for Treating Oropharyngeal Cancer

A 63 year old man is diagnosed with a stage III head and neck cancer. To treat, he receives an intravenous injection of a polypeptide of the present invention (eg. a botulinum type D neurotoxin protease, a botulinum type D neurotoxin translocation domain and a TGF-alpha peptide), optionally in combination with radiotherapy and chemotherapy. Within 4 weeks a significant improvement in the patient's ability to swallow food is apparent which is maintained longer than would be expected with the combination therapy alone.

Example 27—Method for Treating Myeloma Cancer

A 73 year old woman diagnosed with multiple myeloma associated osteolytic bone lesions and hypercalcemia is treated with the standard chemotherapy. To improve the effects of the treatments she receives an intravenous injection of a polypeptide of the present invention (eg. a botulinum type D neurotoxin protease, a botulinum type C neurotoxin translocation domain and an IL-6 peptide). Within 4 weeks a significant reduction in the size of bone lesions and severity of hypercalcemia is observed without appearance of new lesions elsewhere using the usual detection tools (MRI, X-ray, blood tests).

Example 28—Method for Treating Soft Tissue Sarcoma Cancer

A 51 year old woman diagnosed with a fibrosarcoma of the leg is treated with radiation therapy in an attempt to reduce tumour size prior to surgical resection. To improve the effects of the radiation treatment she receives an intravenous injection of a polypeptide of the present invention (eg. a botulinum type D neurotoxin protease, a botulinum type C neurotoxin translocation domain, a GS20 linker, and a bFGF peptide). Within 14 days a significant shrinkage of the tumour over and above that expected with radiation therapy alone is observed, allowing surgical resection with clear margins.

Example 29—Method for Treating Gastric Cancer

A 84 year old man diagnosed with advanced gastric cancer and unable to undergo surgical resection is treated with radiation therapy to relieve tumour associated blockage. To improve the effects of the treatment he receives multiple direct injections of a polypeptide of the present invention (eg. a botulinum type A neurotoxin protease, a botulinum type A neurotoxin translocation domain, a GS20 linker, and a GRP peptide). Within 5 days a significant shrinkage of the tumour is observed with the usual detection tools (gastroscopic examination and CT scan). The treatment is repeated 1 month later and 4 months later tumour blockage has not recurred.

Example 30—Method for Treating Testicular Cancer

A 32 year old man is diagnosed with a stage I seminoma cancer. To treat and/or to prevent recurrence he receives a intravenous injection of a polypeptide of the present invention (eg. a botulinum type D neurotoxin protease, a botulinum type C neurotoxin translocation domain, a GS20 linker, and a VEGF peptide), optionally in combination with chemotherapy. Within 14 days a significant shrinkage of the tumour is observed. The treatment is repeated 1 month later and 6 weeks later no tumour is observable anymore with the usual detection tools (blood tests and CT scans).

Example 31—Method for Treating Uterine Cancer

A 76 year old woman diagnosed with a stage IIA endometrial cancer is treated with usual chemotherapy and radiotherapy. To improve the effects of the treatment and to prevent metastasis she receives a direct injection of a polypeptide of the present invention (eg. a botulinum type A neurotoxin protease and translocation domain and an EGF peptide). Within 6 weeks a significant shrinkage of the tumour is observed on direct visualization of the uterine cavity by hysteroscopy without appearance of metastasis elsewhere.

Example 32—Method for Treating Karposi Sarcoma

A 48 year old man is diagnosed with acquired immunity deficiency syndrome (AIDS) and presents with multiple Karposi sarcoma lesions. To treat, he receives an intravenous injection of a polypeptide of the present invention (eg. a botulinum type A neurotoxin protease, a botulinum type A neurotoxin translocation domain and an IL-6 peptide), optionally in combination with interferon alpha. Within 3 weeks a significant reduction in lesion size is observed without appearance of new lesions elsewhere. The treatment is repeated twice after 1 month and 3 months which effectively stops the progression of the Kaposi sarcoma.

Example 33—Method for Treating Primary Brain Cancer

A 45 year old woman is diagnosed with glioblastoma. To treat and/or to prevent further metastasis she receives an intracranial application of a polypeptide of the present invention (eg. a botulinum type C neurotoxin protease, a botulinum type C neurotoxin translocation domain and a VEGF peptide), optionally in combination with chemotherapy. Within 4 weeks a significant decrease in the size of the tumour is observed without appearance of metastasis elsewhere. The treatment is repeated 1 month later and 4 weeks later no tumour is observable anymore with the usual detection tools (X-rays, CT scan, etc.).

Example 34—Method for Treating Rectal Cancer

A 77 year old man diagnosed with a stage II rectal cancer is treated with a polypeptide of the present invention (eg. botulinum type A neurotoxin protease and an anti-EGFR antibody F(ab)′₂ fragment), optionally incombination with radiotherapy and surgery. He receives localised injections of polypeptide (eg. 3 days in advance of a standard regiment of fractionated radiation) and within 2 weeks a significant shrinkage of the tumour is observed which enables complete surgical resection. 12 months later no tumour recurrence is observable with the usual detection tools (colonoscopy, CT scan, PET scan, etc.) and the level of carcinoembryonic antigen (CEA) returned to the normal.

Example 35—Assessment of Proliferation Changes, Inhibition of Cellular Secretion and Concomitant SNARE Cleavage after Treatment of In Vitro Cultured Renal Cancer Cell Lines with a Polypeptide of the Present Invention Methods: Proliferation Analysis:

An appropriate volume containing 1000 cells of a suitable renal cancer cell line, for example A498, ACHN or 786-0, are seeded into the wells of a 96-well cell culture plate in a suitable growth medium supplemented with 10% Foetal Bovine Serum and incubated at 37° C. in a humidified atmosphere with 5% CO₂. Cells are allowed to adhere overnight after which treatment with an EGF-liganded LHA molecule, such as an LHA molecule with a C-terminal presented EGF ligand, is initiated. After 24 hours, the treatment media are removed, cell monolayers are washed to remove traces of LHA-EGF and fresh medium applied to cells. After a further 24 hours a conventional calorimetric proliferation assay (based on the determination of the cleavage of the tetrazolium salt WST-1 to formazan by cellular enzymes) is performed. Specifically, WST-1 is added to the culture medium for 4 hours after which the optical density at 440 nm is determined for each treatment. FIGS. 50-52 demonstrate inhibition of in vitro proliferation from a renal cell carcinoma line by an EGF-LHA fusion.

Cellular Secretion Analysis:

10,000 cells of a suitable renal cancer cell line (for example 786-0, A498 or ACHN) are seeded in the wells of a 24 well plate in an appropriate culture medium containing 10% foetal bovine serum. Plates are incubated overnight in an incubator at 37° C. in a humidified atmosphere with 5% CO₂ to allow cells to adhere. Cell cultures are then treated with an appropriate polypeptide of the present invention which targets a specific receptor on the cells of interest (for the cell lines detailed above an appropriate molecule would be an EGF-liganded LHA as these 3 cell lines all express EGF receptors, FIG. 48). After 24 hours, the treatment medium is removed, cell monolayers refed with fresh culture medium and 24-48 hours later samples of the culture medium are removed to fresh microfuge tubes which are subsequently spun at 1500 rpm for 5 minutes to remove floating cells. The resultant supernatants are removed to fresh tubes and aliquots then used for the quantification of specific analytes (for example VEGF, TNF-alpha) by enzyme-linked immunosorbent assay or ELISA (FIG. 53). An example of analysis of inhibition of a secretion (GRP) from a small cell lung carcinoma cell line is provided in FIG. 54.

Western Blot Analysis to Detect SNARE Cleavage Due to Cellular Uptake

of Polypeptides of the Present Invention: Cell cultures are treated with polypeptides as detailed above for the analysis of cellular secretions. After removal of culture medium for subsequent ELISA analysis, cell monolayers in each well are washed three times with phosphate-buffered saline and protein extracts prepared by cellular lysis in standard Laemmli sample buffer. Cellular protein are separated on 12% SDS-polyacrylamide gels and electrophoretically transferred to a nitrocellulose membrane. After blocking of the membrane, primary antisera are used to probe for the SNARE protein of interest and detection of full length and/or cleaved forms is enabled by a peroxidase-conjugated anti-species IgG. In the case of treatment of the renal cell line 786-0 with an LHA-EGF molecule, cleavage of the SNARE SNAP-25 is observed (see FIG. 49).

SEQ ID NOs  1. DNA sequence of LH_(N)/A ggatccATGGAGTTCGTTAACAAACAGTTCAACTATAAAGACCCAGTTAACGGTGTTGACATTGCTTAC ATCAAAATCCCGAACGCTGGCCAGATGCAGCCGGTAAAGGCATTCAAAATCCACAACAAAATCTGGGTT ATCCCGGAACGTGATACCTTTACTAACCCGGAAGAAGGTGACCTGAACCCGCCACCGGAAGCGAAACAG GTGCCGGTATCTTACTATGACTCCACCTACCTGTCTACCGATAACGAAAAGGACAACTACCTGAAAGGT GTTACTAAACTGTTCGAGCGTATTTACTCCACCGACCTGGGCCGTATGCTGCTGACTAGCATCGTTCGC GGTATCCCGTTCTGGGGCGGTTCTACCATCGATACCGAACTGAAAGTAATCGACACTAACTGCATCAAC GTTATTCAGCCGGACGGTTCCTATCGTTCCGAAGAACTGAACCTGGTGATCATCGGCCCGTCTGCTGAT ATCATCCAGTTCGAGTGTCTGAGCTTTGGTCACGAAGTTCTGAACCTCACCCGTAACGGCTACGGTTCC ACTCAGTACATCCGTTTCTCTCCGGACTTCACCTTCGGTTTTGAAGAATCCCTGGAAGTAGACACGAAC CCACTGCTGGGCGCTGGTAAATTCGCAACTGATCCTGCGGTTACCCTGGCTCACGAACTGATTCATGCA GGCCACCGCCTGTACGGTATCGCCATCAATCCGAACCGTGTCTTCAAAGTTAACACCAACGCGTATTAC GAGATGTCCGGTCTGGAAGTTAGCTTCGAAGAACTGCGTACTTTTGGCGGTCACGACGCTAAATTCATC GACTCTCTGCAAGAAAACGAGTTCCGTCTGTACTACTATAACAAGTTCAAAGATATCGCATCCACCCTG AACAAAGCGAAATCCATCGTGGGTACCACTGCTTCTCTCCAGTACATGAAGAACGTTTTTAAAGAAAAA TACCTGCTCAGCGAAGACACCTCCGGCAAATTCTCTGTAGACAAGTTGAAATTCGATAAACTTTACAAA ATGCTGACTGAAATTTACACCGAAGACAACTTCGTTAAGTTCTTTAAAGTTCTGAACCGCAAAACCTAT CTGAACTTCGACAAGGCAGTATTCAAAATCAACATCGTGCCGAAAGTTAACTACACTATCTACGATGGT TTCAACCTGCGTAACACCAACCTGGCTGCTAATTTTAACGGCCAGAACACGGAAATCAACAACATGAAC TTCACAAAACTGAAAAACTTCACTGGTCTGTTCGAGTTTTACAAGCTGCTGTGCGTCGACGGCATCATT ACCTCCAAAACTAAATCTGACGATGACGATAAAAACAAAGCGCTGAACCTGCAGTGTATCAAGGTTAAC AACTGGGATTTATTCTTCAGCCCGAGTGAAGACAACTTCACCAACGACCTGAACAAAGGTGAAGAAATC ACCTCAGATACTAACATCGAAGCAGCCGAAGAAAACATCTCGCTGGACCTGATCCAGCAGTACTACCTG ACCTTTAATTTCGACAACGAGCCGGAAAACATTTCTATCGAAAACCTGAGCTCTGATATCATCGGCCAG CTGGAACTGATGCCGAACATCGAACGTTTCCCAAACGGTAAAAAGTACGAGCTGGACAAATATACCATG TTCCACTACCTGCGCGCGCAGGAATTTGAACACGGCAAATCCCGTATCGCACTGACTAACTCCGTTAAC GAAGCTCTGCTCAACCCGTCCCGTGTATACACCTTCTTCTCTAGCGACTACGTGAAAAAGGTCAACAAA GCGACTGAAGCTGCAATGTTCTTGGGTTGGGTTGAACAGCTTGTTTATGATTTTACCGACGAGACGTCC GAAGTATCTACTACCGACAAAATTGCGGATATCACTATCATCATCCCGTACATCGGTCCGGCTCTGAAC ATTGGCAACATGCTGTACAAAGACGACTTCGTTGGCGCACTGATCTTCTCCGGTGCGGTGATCCTGCTG GAGTTCATCCCGGAAATCGCCATCCCGGTACTGGGCACCTTTGCTCTGGTTTCTTACATTGCAAACAAG GTTCTGACTGTACAAACCATCGACAACGCGCTGAGCAAACGTAACGAAAAATGGGATGAAGTTTACAAA TATATCGTGACCAACTGGCTGGCTAAGGTTAATACTCAGATCGACCTCATCCGCAAAAAAATGAAAGAA GCACTGGAAAACCAGGCGGAAGCTACCAAGGCAATCATTAACTACCAGTACAACCAGTACACCGAGGAA GAAAAAAACAACATCAACTTCAACATCGACGATCTGTCCTCTAAACTGAACGAATCCATCAACAAAGCT ATGATCAACATCAACAAGTTCCTGAACCAGTGCTCTGTAAGCTATCTGATGAACTCCATGATCCCGTAC GGTGTTAAACGTCTGGAGGACTTCGATGCGTCTCTGAAAGACGCCCTGCTGAAATACATTTACGACAAC CGTGGCACTCTGATCGGTCAGGTTGATCGTCTGAAGGACAAAGTGAACAATACCTTATCGACCGACATC CCTTTTCAGCTCAGTAAATATGTCGATAACCAACGCCTTTTGTCCACTctagaataatgaaagctt 2. DNA sequence of LH_(N)/B GGATCCATGCCGGTTACCATCAACAACTTCAACTACAACGACCCGATCGACAACAACAACATCATTATG ATGGAACCGCCGTTCGCACGTGGTACCGGACGTTACTACAAGGCTTTTAAGATCACCGACCGTATCTGG ATCATCCCGGAACGTTACACCTTCGGTTACAAACCTGAGGACTTCAACAAGAGTAGCGGGATTTTCAAT CGTGACGTCTGCGAGTACTATGATCCAGATTATCTGAATACCAACGATAAGAAGAACATATTCCTTCAG ACTATGATTAAACTCTTCAACCGTATCAAAAGCAAACCGCTCGGTGAAAAACTCCTCGAAATGATTATC AACGGTATCCCGTACCTCGGTGACCGTCGTGTCCCGCTTGAAGAGTTCAACACCAACATCGCAAGCGTC ACCGTCAACAAACTCATCAGCAACCCAGGTGAAGTCGAACGTAAAAAAGGTATCTTCGCAAACCTCATC ATCTTCGGTCCGGGTCCGGTCCTCAACGAAAACGAAACCATCGACATCGGTATCCAGAACCACTTCGCA AGCCGTGAAGGTTTCGGTGGTATCATGCAGATGAAATTCTGCCCGGAATACGTCAGTGTCTTCAACAAC GTCCAGGAAAACAAAGGTGCAAGCATCTTCAACCGTCGTGGTTACTTCAGCGACCCGGCACTCATCCTC ATGCATGAACTCATCCACGTCCTCCACGGTCTCTACGGTATCAAAGTTGACGACCTCCCGATCGTCCCG AACGAGAAGAAATTCTTCATGCAGAGCACCGACGCAATCCAGGCTGAGGAACTCTACACCTTCGGTGGC CAAGACCCAAGTATCATAACCCCGTCCACCGACAAAAGCATCTACGACAAAGTCCTCCAGAACTTCAGG GGTATCGTGGACAGACTCAACAAAGTCCTCGTCTGCATCAGCGACCCGAACATCAATATCAACATATAC AAGAACAAGTTCAAAGACAAGTACAAATTCGTCGAGGACAGCGAAGGCAAATACAGCATCGACGTAGAA AGTTTCGACAAGCTCTACAAAAGCCTCATGTTCGGTTTCACCGAAACCAACATCGCCGAGAACTACAAG ATCAAGACAAGGGCAAGTTACTTCAGCGACAGCCTCCCGCCTGTCAAAATCAAGAACCTCTTAGACAAC GAGATTTACACAATTGAAGAGGGCTTCAACATCAGTGACAAAGACATGGAGAAGGAATACAGAGGTCAG AACAAGGCTATCAACAAACAGGCATACGAGGAGATCAGCAAAGAACACCTCGCAGTCTACAAGATCCAG ATGTGCGTCGACGGCATCATTACCTCCAAAACTAAATCTGACGATGACGATAAAAACAAAGCGCTGAAC CTGCAGTGCATCGACGTTGACAACGAAGACCTGTTCTTCATCGCTGACAAAAACAGCTTCAGTGACGAC CTGAGCAAAAACGAACGTATCGAATACAACACCCAGAGCAACTACATCGAAAACGACTTCCCGATCAAC GAACTGATCCTGGACACCGACCTGATAAGTAAAATCGAACTGCCGAGCGAAAACACCGAAAGTCTGACC GACTTCAACGTTGACGTTCCGGTTTACGAAAAACAGCCGGCTATCAAGAAAATCTTCACCGACGAAAAC ACCATCTTCCAGTACCTGTACAGCCAGACCTTCCCGCTGGACATCCGTGACATCAGTCTGACCAGCAGT TTCGACGACGCTCTGCTGTTCAGCAACAAAGTTTACAGTTTCTTCAGCATGGACTACATCAAAACCGCT AACAAAGTTGTTGAAGCAGGGCTGTTCGCTGGTTGGGTTAAACAGATCGTTAACGACTTCGTTATCGAA GCTAACAAAAGCAACACTATGGACAAAATCGCTGACATCAGTCTGATCGTTCCGTACATCGGTCTGGCT CTGAACGTTGGTAACGAAACCGCTAAAGGTAACTTTGAAAACGCTTTCGAGATCGCTGGTGCAAGCATC CTGCTGGAGTTCATCCCGGAACTGCTGATCCCGGTTGTTGGTGCTTTCCTGCTGGAAAGTTACATCGAC AACAAAAACAAGATCATCAAAACCATCGACAACGCTCTGACCAAACGTAACGAAAAATGGAGTGATATG TACGGTCTGATCGTTGCTCAGTGGCTGAGCACCGTCAACACCCAGTTCTACACCATCAAAGAAGGTATG TACAAAGCTCTGAACTACCAGGCTCAGGCTCTGGAAGAGATCATCAAATACCGTTACAACATCTACAGT GAGAAGGAAAAGAGTAACATCAACATCGACTTCAACGACATCAACAGCAAACTGAACGAAGGTATCAAC CAGGCTATCGACAACATCAACAACTTCATCAACGGTTGCAGTGTTAGCTACCTGATGAAGAAGATGATC CCGCTGGCTGTTGAAAAACTGCTGGACTTCGACAACACCCTGAAAAAGAACCTGCTGAACTACATCGAC GAAAACAAGCTGTACCTGATCGGTAGTGCTGAATACGAAAAAAGTAAAGTGAACAAATACCTGAAGACC ATCATGCCGTTCGACCTGAGTATCTACACCAACGACACCATCCTGATCGAAATGTTCAACAAATACAAC TCtctagaataatgaaagctt 3. DNA sequence of LH_(N)/C ggatccATGCCGATCACCATCAACAACTTCAACTACAGCGATCCGGTGGATAACAAAAACATCCTGTAC CTGGATACCCATCTGAATACCCTGGCGAACGAACCGGAAAAAGCGTTTCGTATCACCGGCAACATTTGG GTTATTCCGGATCGTTTTAGCCGTAACAGCAACCCGAATCTGAATAAACCGCCGCGTGTTACCAGCCCG AAAAGCGGTTATTACGATCCGAACTATCTGAGCACCGATAGCGATAAAGATACCTTCCTGAAAGAAATC ATCAAACTGTTCAAACGCATCAACAGCCGTGAAATTGGCGAAGAACTGATCTATCGCCTGAGCACCGAT ATTCCGTTTCCGGGCAACAACAACACCCCGATCAACACCTTTGATTTCGATGTGGATTTCAACAGCGTT GATGTTAAAACCCGCCAGGGTAACAATTGGGTGAAAACCGGCAGCATTAACCCGAGCGTGATTATTACC GGTCCGCGCGAAAACATTATTGATCCGGAAACCAGCACCTTTAAACTGACCAACAACACCTTTGCGGCG CAGGAAGGTTTTGGCGCGCTGAGCATTATTAGCATTAGCCCGCGCTTTATGCTGACCTATAGCAACGCG ACCAACGATGTTGGTGAAGGCCGTTTCAGCAAAAGCGAATTTTGCATGGACCCGATCCTGATCCTGATG CATGAACTGAACCATGCGATGCATAACCTGTATGGCATCGCGATTCCGAACGATCAGACCATTAGCAGC GTGACCAGCAACATCTTTTACAGCCAGTACAACGTGAAACTGGAATATGCGGAAATCTATGCGTTTGGC GGTCCGACCATTGATCTGATTCCGAAAAGCGCGCGCAAATACTTCGAAGAAAAAGCGCTGGATTACTAT CGCAGCATTGCGAAACGTCTGAACAGCATTACCACCGCGAATCCGAGCAGCTTCAACAAATATATCGGC GAATATAAACAGAAACTGATCCGCAAATATCGCTTTGTGGTGGAAAGCAGCGGCGAAGTTACCGTTAAC CGCAATAAATTCGTGGAACTGTACAACGAACTGACCCAGATCTTCACCGAATTTAACTATGCGAAAATC TATAACGTGCAGAACCGTAAAATCTACCTGAGCAACGTGTATACCCCGGTGACCGCGAATATTCTGGAT GATAACGTGTACGATATCCAGAACGGCTTTAACATCCCGAAAAGCAACCTGAACGTTCTGTTTATGGGC CAGAACCTGAGCCGTAATCCGGCGCTGCGTAAAGTGAACCCGGAAAACATGCTGTACCTGTTCACCAAA TTTTGCGTCGACGCGATTGATGGTCGTAGCCTGTACAACAAAACCCTGCAGTGTCGTGAACTGCTGGTG AAAAACACCGATCTGCCGTTTATTGGCGATATCAGCGATGTGAAAACCGATATCTTCCTGCGCAAAGAT ATCAACGAAGAAACCGAAGTGATCTACTACCCGGATAACGTGAGCGTTGATCAGGTGATCCTGAGCAAA AACACCAGCGAACATGGTCAGCTGGATCTGCTGTATCCGAGCATTGATAGCGAAAGCGAAATTCTGCCG GGCGAAAACCAGGTGTTTTACGATAACCGTACCCAGAACGTGGATTACCTGAACAGCTATTACTACCTG GAAAGCCAGAAACTGAGCGATAACGTGGAAGATTTTACCTTTACCCGCAGCATTGAAGAAGCGCTGGAT AACAGCGCGAAAGTTTACACCTATTTTCCGACCCTGGCGAACAAAGTTAATGCGGGTGTTCAGGGCGGT CTGTTTCTGATGTGGGCGAACGATGTGGTGGAAGATTTCACCACCAACATCCTGCGTAAAGATACCCTG GATAAAATCAGCGATGTTAGCGCGATTATTCCGTATATTGGTCCGGCGCTGAACATTAGCAATAGCGTG CGTCGTGGCAATTTTACCGAAGCGTTTGCGGTTACCGGTGTGACCATTCTGCTGGAAGCGTTTCCGGAA TTTACCATTCCGGCGCTGGGTGCGTTTGTGATCTATAGCAAAGTGCAGGAACGCAACGAAATCATCAAA ACCATCGATAACTGCCTGGAACAGCGTATTAAACGCTGGAAAGATAGCTATGAATGGATGATGGGCACC TGGCTGAGCCGTATTATCACCCAGTTCAACAACATCAGCTACCAGATGTACGATAGCCTGAACTATCAG GCGGGTGCGATTAAAGCGAAAATCGATCTGGAATACAAAAAATACAGCGGCAGCGATAAAGAAAACATC AAAAGCCAGGTTGAAAACCTGAAAAACAGCCTGGATGTGAAAATTAGCGAAGCGATGAATAACATCAAC AAATTCATCCGCGAATGCAGCGTGACCTACCTGTTCAAAAACATGCTGCCGAAAGTGATCGATGAACTG AACGAATTTGATCGCAACACCAAAGCGAAACTGATCAACCTGATCGATAGCCACAACATTATTCTGGTG GGCGAAGTGGATAAACTGAAAGCGAAAGTTAACAACAGCTTCCAGAACACCATCCCGTTTAACATCTTC AGCTATACCAACAACAGCCTGCTGAAAGATATCATCAACGAATACTTCAAtctagaataatgaaagctt 4. DNA sequence of LH_(N)/D ggatccATGACGTGGCCAGTTAAGGATTTCAACTACTCAGATCCTGTAAATGACAACGATATTCTGTAC CTTCGCATTCCACAAAATAAACTGATCACCACACCAGTCAAAGCATTCATGATTACTCAAAACATTTGG GTCATTCCAGAACGCTTTTCTAGTGACACAAATCCGAGTTTATCTAAACCTCCGCGTCCGACGTCCAAA TATCAGAGCTATTACGATCCCTCATATCTCAGTACGGACGAACAAAAAGATACTTTCCTTAAAGGTATC ATTAAACTGTTTAAGCGTATTAATGAGCGCGATATCGGGAAAAAGTTGATTAATTATCTTGTTGTGGGT TCCCCGTTCATGGGCGATAGCTCTACCCCCGAAGACACTTTTGATTTTACCCGTCATACGACAAACATC GCGGTAGAGAAGTTTGAGAACGGATCGTGGAAAGTCACAAACATCATTACACCTAGCGTCTTAATTTTT GGTCCGCTGCCAAACATCTTAGATTATACAGCCAGCCTGACTTTGCAGGGGCAACAGTCGAATCCGAGT TTCGAAGGTTTTGGTACCCTGAGCATTCTGAAAGTTGCCCCGGAATTTCTGCTCACTTTTTCAGATGTC ACCAGCAACCAGAGCTCAGCAGTATTAGGAAAGTCAATTTTTTGCATGGACCCGGTTATTGCACTGATG CACGAACTGACGCACTCTCTGCATCAACTGTATGGGATCAACATCCCCAGTGACAAACGTATTCGTCCC CAGGTGTCTGAAGGATTTTTCTCACAGGATGGGCCGAACGTCCAGTTCGAAGAGTTGTATACTTTCGGA GGCCTGGACGTAGAGATCATTCCCCAGATTGAGCGCAGTCAGCTGCGTGAGAAGGCATTGGGCCATTAT AAGGATATTGCAAAACGCCTGAATAACATTAACAAAACGATTCCATCTTCGTGGATCTCGAATATTGAT AAATATAAGAAAATTTTTAGCGAGAAATATAATTTTGATAAAGATAATACAGGTAACTTTGTGGTTAAC ATTGACAAATTCAACTCCCTTTACAGTGATTTGACGAATGTAATGAGCGAAGTTGTGTATAGTTCCCAA TACAACGTTAAGAATCGTACCCATTACTTCTCTCGTCACTACCTGCCGGTTTTCGCGAACATCCTTGAC GATAATATTTACACTATTCGTGACGGCTTTAACTTGACCAACAAGGGCTTCAATATTGAAAATTCAGGC CAGAACATTGAACGCAACCCGGCCTTGCAGAAACTGTCGAGTGAATCCGTGGTTGACCTGTTTACCAAA GTCTGCGTCGACAAAAGCGAAGAGAAGCTGTACGATGACGATGACAAAGATCGTTGGGGATCGTCCCTG CAGTGTATTAAAGTGAAAAACAATCGGCTGCCTTATGTAGCAGATAAAGATAGCATTAGTCAGGAGATT TTCGAAAATAAAATTATCACTGACGAAACCAATGTTCAGAATTATTCAGATAAATTTTCACTGGACGAA AGCATCTTAGATGGCCAAGTTCCGATTAACCCGGAAATTGTTGATCCGTTACTGCCGAACGTGAATATG GAACCGTTAAACCTCCCTGGCGAAGAGATCGTATTTTATGATGACATTACGAAATATGTGGACTACCTT AATTCTTATTACTATTTGGAAAGCCAGAAACTGTCCAATAACGTGGAAAACATTACTCTGACCACAAGC GTGGAAGAGGCTTTAGGCTACTCAAATAAGATTTATACCTTCCTCCCGTCGCTGGCGGAAAAAGTAAAT AAAGGTGTGCAGGCTGGTCTGTTCCTCAACTGGGCGAATGAAGTTGTCGAAGACTTTACCACGAATATT ATGAAAAAGGATACCCTGGATAAAATCTCCGACGTCTCGGTTATTATCCCATATATTGGCCCTGCGTTA AATATCGGTAATAGTGCGCTGCGGGGGAATTTTAACCAGGCCTTTGCTACCGCGGGCGTCGCGTTCCTC CTGGAGGGCTTTCCTGAATTTACTATCCCGGCGCTCGGTGTTTTTACATTTTACTCTTCCATCCAGGAG CGTGAGAAAATTATCAAAACCATCGAAAACTGCCTGGAGCAGCGGGTGAAACGCTGGAAAGATTCTTAT CAATGGATGGTGTCAAACTGGTTATCTCGCATCACGACCCAATTCAACCATATTAATTACCAGATGTAT GATAGTCTGTCGTACCAAGCTGACGCCATTAAAGCCAAAATTGATCTGGAATATAAAAAGTACTCTGGT AGCGATAAGGAGAACATCAAAAGCCAGGTGGAGAACCTTAAGAATAGTCTGGATGTGAAAATCTCTGAA GCTATGAATAACATTAACAAATTCATTCGTGAATGTTCGGTGACGTACCTGTTCAAGAATATGCTGCCA AAAGTTATTGATGAACTGAATAAATTTGATCTGCGTACCAAAACCGAACTTATCAACCTCATCGACTCC CACAACATTATCCTTGTGGGCGAAGTGGATCGTCTGAAGGCCAAAGTAAACGAGAGCTTTGAAAATACG ATGCCGTTTAATATTTTTTCATATACCAATAACTCCTTGCTGAAAGATATCATCAATGAATATTTCAAT ctagattaataagctt 5. DNA sequence of the CT-CST29 linker GGATCCGTCGACCTGCAGGGTCTAGAAGGCGGTGGCGGTAGCGGCGGTGGCGGTAGCGGCGGTGGCGGT AGCGGCGGTGGCGGTAGCGCACTAGTGCAGGAAAGACCTCCATTACAACAACCTCCACATCGCGATAAG AAACCATGTAAGAATTTCTTTTGGAAAACATTTAGCAGTTGCAAATGATAAAAGCTT 6. DNA sequence of the LHD-CT-CST29 fusion GGATCCATGACGTGGCCAGTTAAGGATTTCAACTACTCAGATCCTGTAAATGACAACGATATTCTGTAC CTTCGCATTCCACAAAATAAACTGATCACCACACCAGTCAAAGCATTCATGATTACTCAAAACATTTGG GTCATTCCAGAACGCTTTTCTAGTGACACAAATCCGAGTTTATCTAAACCTCCGCGTCCGACGTCCAAA TATCAGAGCTATTACGATCCCTCATATCTCAGTACGGACGAACAAAAAGATACTTTCCTTAAAGGTATC ATTAAACTGTTTAAGCGTATTAATGAGCGCGATATCGGGAAAAAGTTGATTAATTATCTTGTTGTGGGT TCCCCGTTCATGGGCGATAGCTCTACCCCCGAAGACACTTTTGATTTTACCCGTCATACGACAAACATC GCGGTAGAGAAGTTTGAGAACGGATCGTGGAAAGTCACAAACATCATTACACCTAGCGTCTTAATTTTT GGTCCGCTGCCAAACATCTTAGATTATACAGCCAGCCTGACTTTGCAGGGGCAACAGTCGAATCCGAGT TTCGAAGGTTTTGGTACCCTGAGCATTCTGAAAGTTGCCCCGGAATTTCTGCTCACTTTTTCAGATGTC ACCAGCAACCAGAGCTCAGCAGTATTAGGAAAGTCAATTTTTTGCATGGACCCGGTTATTGCACTGATG CACGAACTGACGCACTCTCTGCATCAACTGTATGGGATCAACATCCCCAGTGACAAACGTATTCGTCCC CAGGTGTCTGAAGGATTTTTCTCACAGGATGGGCCGAACGTCCAGTTCGAAGAGTTGTATACTTTCGGA GGCCTGGACGTAGAGATCATTCCCCAGATTGAGCGCAGTCAGCTGCGTGAGAAGGCATTGGGCCATTAT AAGGATATTGCAAAACGCCTGAATAACATTAACAAAACGATTCCATCTTCGTGGATCTCGAATATTGAT AAATATAAGAAAATTTTTAGCGAGAAATATAATTTTGATAAAGATAATACAGGTAACTTTGTGGTTAAC ATTGACAAATTCAACTCCCTTTACAGTGATTTGACGAATGTAATGAGCGAAGTTGTGTATAGTTCCCAA TACAACGTTAAGAATCGTACCCATTACTTCTCTCGTCACTACCTGCCGGTTTTCGCGAACATCCTTGAC GATAATATTTACACTATTCGTGACGGCTTTAACTTGACCAACAAGGGCTTCAATATTGAAAATTCAGGC CAGAACATTGAACGCAACCCGGCCTTGCAGAAACTGTCGAGTGAATCCGTGGTTGACCTGTTTACCAAA GTCTGCGTCGACAAAAGCGAAGAGAAGCTGTACGATGACGATGACAAAGATCGTTGGGGATCGTCCCTG CAGTGTATTAAAGTGAAAAACAATCGGCTGCCTTATGTAGCAGATAAAGATAGCATTAGTCAGGAGATT TTCGAAAATAAAATTATCACTGACGAAACCAATGTTCAGAATTATTCAGATAAATTTTCACTGGACGAA AGCATCTTAGATGGCCAAGTTCCGATTAACCCGGAAATTGTTGATCCGTTACTGCCGAACGTGAATATG GAACCGTTAAACCTCCCTGGCGAAGAGATCGTATTTTATGATGACATTACGAAATATGTGGACTACCTT AATTCTTATTACTATTTGGAAAGCCAGAAACTGTCCAATAACGTGGAAAACATTACTCTGACCACAAGC GTGGAAGAGGCTTTAGGCTACTCAAATAAGATTTATACCTTCCTCCCGTCGCTGGCGGAAAAAGTAAAT AAAGGTGTGCAGGCTGGTCTGTTCCTCAACTGGGCGAATGAAGTTGTCGAAGACTTTACCACGAATATT ATGAAAAAGGATACCCTGGATAAAATCTCCGACGTCTCGGTTATTATCCCATATATTGGCCCTGCGTTA AATATCGGTAATAGTGCGCTGCGGGGGAATTTTAACCAGGCCTTTGCTACCGCGGGCGTCGCGTTCCTC CTGGAGGGCTTTCCTGAATTTACTATCCCGGCGCTCGGTGTTTTTACATTTTACTCTTCCATCCAGGAG CGTGAGAAAATTATCAAAACCATCGAAAACTGCCTGGAGCAGCGGGTGAAACGCTGGAAAGATTCTTAT CAATGGATGGTGTCAAACTGGTTATCTCGCATCACGACCCAATTCAACCATATTAATTACCAGATGTAT GATAGTCTGTCGTACCAAGCTGACGCCATTAAAGCCAAAATTGATCTGGAATATAAAAAGTACTCTGGT AGCGATAAGGAGAACATCAAAAGCCAGGTGGAGAACCTTAAGAATAGTCTGGATGTGAAAATCTCTGAA GCTATGAATAACATTAACAAATTCATTCGTGAATGTTCGGTGACGTACCTGTTCAAGAATATGCTGCCA AAAGTTATTGATGAACTGAATAAATTTGATCTGCGTACCAAAACCGAACTTATCAACCTCATCGACTCC CACAACATTATCCTTGTGGGCGAAGTGGATCGTCTGAAGGCCAAAGTAAACGAGAGCTTTGAAAATACG ATGCCGTTTAATATTTTTTCATATACCAATAACTCCTTGCTGAAAGATATCATCAATGAATATTTCAAT CTAGAAGGCGGTGGCGGTAGCGGCGGTGGCGGTAGCGGCGGTGGCGGTAGCGCACTAGTGCAGGAAAGA CCTCCATTACAACAACCTCCACATCGCGATAAGAAACCATGTAAGAATTTCTTTTGGAAAACATTTAGC AGTTGCAAAtaataagctt 7. Protein sequence of the LHD-CT-CST29 fusion TWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQS YYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVE KFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSN QSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLD VEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDK FNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNI ERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFEN KIITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSY YYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKK DTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREK IIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDK ENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNI ILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALVQERPPL QQPPHRDKKPCKNFFWKTFSSCK 8. DNA sequence of the CP-EGF linker ggatccGTCGACaacaacaataacaacaacaataacaacaacgacgatgacgataaaAATTCAGATAGC GAATGTCCACTTAGTCACGACGGGTACTGTTTGCATGATGGTGTGTGTATGTATATAGAAGCACTAGAC AAATACGCTTGCAATTGCGTAGTTGGCTATATAGGAGAGCGATGCCAATATAGAGATCTGAAGTGGTGG GAGTTAAGGGCAgaagcggcagccaaagaagcagccgctaaggcgctgcagagtctagaataataagct t 9. DNA sequence of the LHA-CP-EGF fusion ggatccATGGAGTTCGTTAACAAACAGTTCAACTATAAAGACCCAGTTAACGGTGTTGACATTGCTTAC ATCAAAATCCCGAACGCTGGCCAGATGCAGCCGGTAAAGGCATTCAAAATCCACAACAAAATCTGGGTT ATCCCGGAACGTGATACCTTTACTAACCCGGAAGAAGGTGACCTGAACCCGCCACCGGAAGCGAAACAG GTGCCGGTATCTTACTATGACTCCACCTACCTGTCTACCGATAACGAAAAGGACAACTACCTGAAAGGT GTTACTAAACTGTTCGAGCGTATTTACTCCACCGACCTGGGCCGTATGCTGCTGACTAGCATCGTTCGC GGTATCCCGTTCTGGGGCGGTTCTACCATCGATACCGAACTGAAAGTAATCGACACTAACTGCATCAAC GTTATTCAGCCGGACGGTTCCTATCGTTCCGAAGAACTGAACCTGGTGATCATCGGCCCGTCTGCTGAT ATCATCCAGTTCGAGTGTCTGAGCTTTGGTCACGAAGTTCTGAACCTCACCCGTAACGGCTACGGTTCC ACTCAGTACATCCGTTTCTCTCCGGACTTCACCTTCGGTTTTGAAGAATCCCTGGAAGTAGACACGAAC CCACTGCTGGGCGCTGGTAAATTCGCAACTGATCCTGCGGTTACCCTGGCTCACGAACTGATTCATGCA GGCCACCGCCTGTACGGTATCGCCATCAATCCGAACCGTGTCTTCAAAGTTAACACCAACGCGTATTAC GAGATGTCCGGTCTGGAAGTTAGCTTCGAAGAACTGCGTACTTTTGGCGGTCACGACGCTAAATTCATC GACTCTCTGCAAGAAAACGAGTTCCGTCTGTACTACTATAACAAGTTCAAAGATATCGCATCCACCCTG AACAAAGCGAAATCCATCGTGGGTACCACTGCTTCTCTCCAGTACATGAAGAACGTTTTTAAAGAAAAA TACCTGCTCAGCGAAGACACCTCCGGCAAATTCTCTGTAGACAAGTTGAAATTCGATAAACTTTACAAA ATGCTGACTGAAATTTACACCGAAGACAACTTCGTTAAGTTCTTTAAAGTTCTGAACCGCAAAACCTAT CTGAACTTCGACAAGGCAGTATTCAAAATCAACATCGTGCCGAAAGTTAACTACACTATCTACGATGGT TTCAACCTGCGTAACACCAACCTGGCTGCTAATTTTAACGGCCAGAACACGGAAATCAACAACATGAAC TTCACAAAACTGAAAAACTTCACTGGTCTGTTCGAGTTTTACAAGCTGCTGTGCGTCGACaacaacaat aacaacaacaataacaacaacgacgatgacgataaaAATTCAGATAGCGAATGTCCACTTAGTCACGAC GGGTACTGTTTGCATGATGGTGTGTGTATGTATATAGAAGCACTAGACAAATACGCTTGCAATTGCGTA GTTGGCTATATAGGAGAGCGATGCCAATATAGAGATCTGAAGTGGTGGGAGTTAAGGGCAgaagcggca gccaaagaagcagccgctaaggcgCTGCAGTGTATCAAGGTTAACAACTGGGATTTATTCTTCAGCCCG AGTGAAGACAACTTCACCAACGACCTGAACAAAGGTGAAGAAATCACCTCAGATACTAACATCGAAGCA GCCGAAGAAAACATCTCGCTGGACCTGATCCAGCAGTACTACCTGACCTTTAATTTCGACAACGAGCCG GAAAACATTTCTATCGAAAACCTGAGCTCTGATATCATCGGCCAGCTGGAACTGATGCCGAACATCGAA CGTTTCCCAAACGGTAAAAAGTACGAGCTGGACAAATATACCATGTTCCACTACCTGCGCGCGCAGGAA TTTGAACACGGCAAATCCCGTATCGCACTGACTAACTCCGTTAACGAAGCTCTGCTCAACCCGTCCCGT GTATACACCTTCTTCTCTAGCGACTACGTGAAAAAGGTCAACAAAGCGACTGAAGCTGCAATGTTCTTG GGTTGGGTTGAACAGCTTGTTTATGATTTTACCGACGAGACGTCCGAAGTATCTACTACCGACAAAATT GCGGATATCACTATCATCATCCCGTACATCGGTCCGGCTCTGAACATTGGCAACATGCTGTACAAAGAC GACTTCGTTGGCGCACTGATCTTCTCCGGTGCGGTGATCCTGCTGGAGTTCATCCCGGAAATCGCCATC CCGGTACTGGGCACCTTTGCTCTGGTTTCTTACATTGCAAACAAGGTTCTGACTGTACAAACCATCGAC AACGCGCTGAGCAAACGTAACGAAAAATGGGATGAAGTTTACAAATATATCGTGACCAACTGGCTGGCT AAGGTTAATACTCAGATCGACCTCATCCGCAAAAAAATGAAAGAAGCACTGGAAAACCAGGCGGAAGCT ACCAAGGCAATCATTAACTACCAGTACAACCAGTACACCGAGGAAGAAAAAAACAACATCAACTTCAAC ATCGACGATCTGTCCTCTAAACTGAACGAATCCATCAACAAAGCTATGATCAACATCAACAAGTTCCTG AACCAGTGCTCTGTAAGCTATCTGATGAACTCCATGATCCCGTACGGTGTTAAACGTCTGGAGGACTTC GATGCGTCTCTGAAAGACGCCCTGCTGAAATACATTTACGACAACCGTGGCACTCTGATCGGTCAGGTT GATCGTCTGAAGGACAAAGTGAACAATACCTTATCGACCGACATCCCTTTTCAGCTCAGTAAATATGTC GATAACCAACGCCTTTTGTCCACTctagaataatgaaagctt 10. Protein sequence of the LHA-CP-EGF fusion EFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPV SYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQ PDGSYRSEELNLVIIGPSADIIQFECLSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLL GAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSL QENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLT EIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTK LKNFTGLFEFYKLLCVDNNNNNNNNNNDDDDKNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGY IGERCQYRDLKWWELRAEAAAKEAAAKALQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEE NISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEH GKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADI TIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNAL SKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDD LSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRL KDKVNNTLSTDIPFQLSKYVDNQRLLST 11. Protein sequence of LH_(N)/A EFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPV SYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQ PDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLL GAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSL QENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLT EIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTK LKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSD TNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHY LRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVS TTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLT VQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKN NINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGT LIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLST 12. Protein sequence of LH_(N)/B PVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDV CEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVN KLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQE NKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDP SIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFD KLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKA INKQAYEEISKEHLAVYKIQMCVDEEKLYDDDDKDRWGSSLQCIDVDNEDLFFIADKNSFSDDLSKNER IEYNTQSNYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYL YSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNT MDAIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKNKII KTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSN INIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYL IGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNS 13. Protein sequence of LH_(N)/C PITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPKSG YYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVK TRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATND VGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPT IDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNK FVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNL SRNPALRKVNPENMLYLFTKFCVDAIDGRSLYNKTLQCRELLVKNTDLPFIGDISDVKTDIFLRKDINE ETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQ KLSDNVEDFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKI SDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTID NCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQ VENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEV DKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFN 14. Protein sequence of LH_(N)/D TWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQS YYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVE KFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSN QSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLD VEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDK FNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNI ERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFEN KIITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSY YYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKK DTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREK IIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDK ENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNI ILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFN 15. Synthesised GnRH peptide pGlu-His-Trp-Ser-Tyr-Gly-Cys-Arg-Pro-Gly-NH2 16. Protein sequence of the LHB-CT-SST28 fusion PVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDV CEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVN KLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQE NKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDP SIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFD KLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKA INKQAYEEISKEHLAVYKIQMCVDGIITSKTKSDDDDKNKALNLQCIDVDNEDLFFIADKNSFSDDLSK NERIEYNTQSNYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIF QYLYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANK SNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKN KIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKE KSNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENK LYLIGSAEYEKSKVNKYLKTIMPFDLSIYINDTILIEMFNKYNSLEGGGGSGGGGSGGGGSALDSANSN PAMAPRERKAGCKNFFWKTFTSC 17. Protein sequence of the LHA-CP-SST28 fusion EFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDIFINPEEGDLNPPPEAKQVPV SYYDSTYLSIDNEKDNYLKGVIKLFERIYSIDLGRMLLTSIVRGIPFWGGSTIDTELKVIDINCINVIQ PDGSYRSEELNLVIIGPSADIIQFECLSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLL GAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRIFGGHDAKFIDSL QENEFRLYYYNKFKDIASTLNKAKSIVGITASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLT EIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNINLAANFNGQNTEINNMNFIK LKNFTGLFEFYKLLCVDGIITSKIKSDDDDKSANSNPAMAPRERKAGCKNFFWKIFTSCALAGGGGSGG GGSGGGGSALVLQCIKVNNWDLFFSPSEDNFINDLNKGEEITSDINIEAAEENISLDLIQQYYLIFNFD NEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALINSVNEALLN PSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFIDETSEVSTIDKIADITIIIPYIGPALNIGNML YKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLIVQTIDNALSKRNEKWDEVYKYIVIN WLAKVNIQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININ KFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGILIGQVDRLKDKVNNTLSTDIPFQLS KYVDNQRLLST 18. Protein sequence of the LHD-CT-EGF fusion TWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDINPSLSKPPRPTSKYQS YYDPSYLSTDEQKDIFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDIFDFTRHTTNIAVE KFENGSWKVINIIIPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLIFSDVISN QSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLD VEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNIGNFVVNIDK FNSLYSDLINVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLINKGFNIENSGQNI ERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFEN KIITDEINVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSY YYLESQKLSNNVENITLITSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKK DILDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFIFYSSIQEREK IIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDK ENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRIKTELINLIDSHNI ILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALVNSDSEC PLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR 19. Protein sequence of the LHD-CT-VIP fusion TWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDINPSLSKPPRPTSKYQS YYDPSYLSTDEQKDIFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDIFDFTRHTTNIAVE KFENGSWKVINIIIPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLIFSDVISN QSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLD VEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNIGNFVVNIDK FNSLYSDLINVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLINKGFNIENSGQNI ERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFEN KIITDEINVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSY YYLESQKLSNNVENITLITSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKK DILDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFIFYSSIQEREK IIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDK ENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRIKTELINLIDSHNI ILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALVHSDAVF TDNYTRLRKQMAVKKYLNSILN 20. Protein sequence of the LHC-CT-IGF1 fusion PITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVISPKSG YYDPNYLSTDSDKDIFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVK TRQGNNWVKIGSINPSVIITGPRENIIDPETSTFKLINNTFAAQEGFGALSIISISPRFMLTYSNAIND VGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVISNIFYSQYNVKLEYAEIYAFGGPT IDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVIVNRNK FVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNL SRNPALRKVNPENMLYLFTKFCVDAIDGRSLYNKTLQCRELLVKNIDLPFIGDISDVKIDIFLRKDINE ETEVIYYPDNVSVDQVILSKNISEHGQLDLLYPSIDSESEILPGENQVFYDNRIQNVDYLNSYYYLESQ KLSDNVEDFIFIRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDILDKI SDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTID NCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQ VENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNIKAKLINLIDSHNIILVGEV DKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALVGPETLCGAELVD ALQFVCGDRGFYFNKPIGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA 21. Protein sequence of the LHD-CT-IGF1 fusion TWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDINPSLSKPPRPTSKYQS YYDPSYLSTDEQKDIFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDIFDFTRHTTNIAVE KFENGSWKVINIIIPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLIFSDVISN QSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLD VEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNIGNFVVNIDK FNSLYSDLINVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLINKGFNIENSGQNI ERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFEN KIITDEINVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSY YYLESQKLSNNVENITLITSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKK DILDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFIFYSSIQEREK IIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDK ENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRIKTELINLIDSHNI ILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALVGPETLC GAELVDALQFVCGDRGFYFNKPIGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA 22. Protein sequence of the LHC-CT-VIP fusion PITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVISPKSG YYDPNYLSTDSDKDIFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVK TRQGNNWVKIGSINPSVIITGPRENIIDPETSTFKLINNTFAAQEGFGALSIISISPRFMLTYSNAIND VGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVISNIFYSQYNVKLEYAEIYAFGGPT IDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVIVNRNK FVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNL SRNPALRKVNPENMLYLFTKFCVDAIDGRSLYNKTLQCRELLVKNIDLPFIGDISDVKIDIFLRKDINE ETEVIYYPDNVSVDQVILSKNISEHGQLDLLYPSIDSESEILPGENQVFYDNRIQNVDYLNSYYYLESQ KLSDNVEDFIFIRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDILDKI SDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTID NCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQ VENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNIKAKLINLIDSHNIILVGEV DKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALVHSDAVFIDNYTR LRKQMAVKKYLNSILN 23. Protein sequence of the LHC-CT-GnRH fusion PITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVISPKSG YYDPNYLSTDSDKDIFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVK TRQGNNWVKIGSINPSVIITGPRENIIDPETSTFKLINNTFAAQEGFGALSIISISPRFMLTYSNAIND VGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVISNIFYSQYNVKLEYAEIYAFGGPT IDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVIVNRNK FVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNL SRNPALRKVNPENMLYLFTKFCVDAIDGRSLYNKTLQCRELLVKNIDLPFIGDISDVKIDIFLRKDINE ETEVIYYPDNVSVDQVILSKNISEHGQLDLLYPSIDSESEILPGENQVFYDNRIQNVDYLNSYYYLESQ KLSDNVEDFIFIRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDILDKI SDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTID NCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQ VENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNIKAKLINLIDSHNIILVGEV DKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALVMKPIQKLLAGLI LLTWCVEGCSSQHWSYGLRPGGKRDAENLIDSFQEIVKEVGQLAETQRFECITHQPRSPLRDLKGALES LIEEETGQKKI 24. Protein sequence of the LHD-CT-GnRH fusion TWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDINPSLSKPPRPTSKYQS YYDPSYLSTDEQKDIFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDIFDFTRHTTNIAVE KFENGSWKVINIIIPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLIFSDVISN QSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLD VEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNIGNFVVNIDK FNSLYSDLINVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLINKGFNIENSGQNI ERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFEN KIITDEINVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSY YYLESQKLSNNVENITLITSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKK DILDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFIFYSSIQEREK IIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDK ENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRIKTELINLIDSHNI ILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSGGGGSALVM KPIQKLLAGLILLTWCVEGCSSQHWSYGLRPGGKRDAENLIDSFQEIVKEVGQLAETQRFECITHQPRS PLRDLKGALESLIEEETGQKKI 25. Protein sequence of the LHD-CT-GRP fusion TWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDINPSLSKPPRPTSKYQS YYDPSYLSTDEQKDIFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDIFDFTRHTTNIAVE KFENGSWKVINIIIPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLIFSDVISN QSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLD VEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNIGNFVVNIDK FNSLYSDLINVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLINKGFNIENSGQNI ERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFEN KIITDEINVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSY YYLESQKLSNNVENITLITSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKK DILDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFIFYSSIQEREK IIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDK ENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRIKTELINLIDSHNI ILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALVGNHWAV GHLM 26. Protein sequence of the LHB-CT-GRP fusion PVTINNFNYNDPIDNNNIIMMEPPFARGIGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDV CEYYDPDYLNINDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNINIASVIVN KLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQE NKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDP SIIIPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFD KLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKA INKQAYEEISKEHLAVYKIQMCVDEEKLYDDDDKDRWGSSLQCIDVDNEDLFFIADKNSFSDDLSKNER IEYNTQSNYIENDFPINELILDIDLISKIELPSENTESLIDFNVDVPVYEKQPAIKKIFTDENTIFQYL YSQTFPLDIRDISLISSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNT MDAIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKNKII KTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSN INIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNILKKNLLNYIDENKLYL IGSAEYEKSKVNKYLKTIMPFDLSITENDTILIEMFNKYNSLEGGGGSGGGGSGGGGSALVGNHWAVGH LM 27. Protein sequence of the LHC-CT-LIF fusion PITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVISPKSG YYDPNYLSTDSDKDIFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVK TRQGNNWVKIGSINPSVIITGPRENIIDPETSTFKLINNTFAAQEGFGALSIISISPRFMLTYSNAIND VGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVISNIFYSQYNVKLEYAEIYAFGGPT IDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVIVNRNK FVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNL SRNPALRKVNPENMLYLFTKFCVDAIDGRSLYNKTLQCRELLVKNIDLPFIGDISDVKIDIFLRKDINE ETEVIYYPDNVSVDQVILSKNISEHGQLDLLYPSIDSESEILPGENQVFYDNRIQNVDYLNSYYYLESQ KLSDNVEDFIFIRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDILDKI SDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTID NCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQ VENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNIKAKLINLIDSHNIILVGEV DKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALVSPLPITPVNATC AIRHPCHNNLMNQIRSQLAQLNGSANALFILYYTAQGEPFPNNLDKLCGPNVIDFPPFHANGTEKAKLV ELYRIVVYLGTSLGNITRDQKILNPSALSLHSKLNATADILRGLLSNVLCRLCSKYHVGHVDVTYGPDT SGKDVFQKKKLGCQLLGKYKQIIAVLAQAF 28. Protein sequence of the LHB-CP-LIF fusion PVTINNFNYNDPIDNNNIIMMEPPFARGIGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDV CEYYDPDYLNINDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNINIASVIVN KLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQE NKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDP SIIIPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFD KLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKA INKQAYEEISKEHLAVYKIQMCVDNNNNNNNNNNDDDDKSPLPITPVNATCAIRHPCHNNLMNQIRSQL AQLNGSANALFILYYTAQGEPFPNNLDKLCGPNVIDFPPFHANGTEKAKLVELYRIVVYLGTSLGNITR DQKILNPSALSLHSKLNATADILRGLLSNVLCRLCSKYHVGHVDVTYGPDTSGKDVFQKKKLGCQLLGK YKQIIAVLAQAFAEAAAKEAAAKALQCIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPI NELILDIDLISKIELPSENTESLIDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTS SFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNIMDKIADISLIVPYIGL ALNVGNETAKGNFENAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSD MYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGI NQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNILKKNLLNYIDENKLYLIGSAEYEKSKVNKYLK TIMPFDLSIYINDTILIEMFNKYNS 29. Protein sequence of the LHC-CT-FGF1 fusion PITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVISPKSG YYDPNYLSTDSDKDIFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVK TRQGNNWVKIGSINPSVIITGPRENIIDPETSTFKLINNTFAAQEGFGALSIISISPRFMLTYSNAIND VGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVISNIFYSQYNVKLEYAEIYAFGGPT IDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVIVNRNK FVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNL SRNPALRKVNPENMLYLFTKFCVDAIDGRSLYNKTLQCRELLVKNIDLPFIGDISDVKIDIFLRKDINE ETEVIYYPDNVSVDQVILSKNISEHGQLDLLYPSIDSESEILPGENQVFYDNRIQNVDYLNSYYYLESQ KLSDNVEDFIFIRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDILDKI SDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTID NCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQ VENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNIKAKLINLIDSHNIILVGEV DKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALVMFNLPPGNYKKP KLLYCSNGGHFLRILPDGIVDGIRDRSDQHIQLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPN EECLFLERLEENHYNTYISKKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPLPVSSD 30. Protein sequence of the LHA-CP-FGF1 fusion EFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDIFINPEEGDLNPPPEAKQVPV SYYDSTYLSIDNEKDNYLKGVIKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDINCINVIQ PDGSYRSEELNLVIIGPSADIIQFECLSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLL GAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRITGGHDAKFIDSL QENEFRLYYYNKFKDIASTLNKAKSIVGITASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLT EIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNINLAANFNGQNTEINNMNFIK LKNFTGLFEFYKLLCVDGIITSKIKSDDDDKMFNLPPGNYKKPKLLYCSNGGHFLRILPDGIVDGIRDR SDQHIQLQLSAESVGEVYIKSTETGQYLAMDITGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEKN WFVGLKKNGSCKRGPRTHYGQKAILFLPLPVSSDGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSE DNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERF PNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGW VEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPV LGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATK AIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDA SLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLST 31. Protein sequence of the LHA-CT-FGF9 fusion EFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPV SYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQ PDGSYRSEELNLVIIGPSADIIQFECLSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLL GAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSL QENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLT EIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTK LKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSD TNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHY LRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVS TTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLT VQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKN NINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGT LIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSGGGGSALVDHLGQSEA GGLPRGPAVTDLDHLKGILRRRQLYCRTGFHLEIFPNGTIQGTRKDHSRFGILEFISIAVGLVSIRGVD SGLYLGMNEKGELYGSEKLTQECVFREQFEENWYNTYSSNLYKHVDTGRRYYVALNKDGTPREGTRTKR HQKFTHFLPRPVDPDKVPELYKDILSQS 32. Protein sequence of the LHC-CP-FGF9 fusion PITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPKSG YYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVK TRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATND VGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPT IDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNK FVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNL SRNPALRKVNPENMLYLFTKFCVDNNNNNNNNNNDDDDKDHLGQSEAGGLPRGPAVTDLDHLKGILRRR QLYCRTGFHLEIFPNGTIQGTRKDHSRFGILEFISIAVGLVSIRGVDSGLYLGMNEKGELYGSEKLTQE CVFREQFEENWYNTYSSNLYKHVDTGRRYYVALNKDGTPREGTRTKRHQKFTHFLPRPVDPDKVPELYK DILSQSAEAAAKEAAAKALQCRELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVI LSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEE ALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNIS NSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWM MGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMN NINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTIPF NIFSYTNNSLLKDIINEYFN 33. Protein sequence of the IgA-HNtet-CT-SST14 Fusion ESNQPEKNGTATKPENSGNTTSENGQTEPEKKLELRNVSDIELYSQTNGTYRQHVSLDGIPENTDTYFV KVKSSAFKDVYIPVASITEEKRNGQSVYKITAKAEKLQQELENKYVDNFTFYLDKKAKEENTNFTSFSN LVKAINQNPSGTYHLAASLNANEVELGPDERSYIKDTFTGRLIGEKDGKNYAIYNLKKPLFENLSGATV EKLSLKNVAISGKNDIGSLANEATNGTKIKQVHVDGCVDGIITSKTKSDDDDKNKALNLQCIKIKNEDL TFIAEKNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSKITLPNDRTTPVTKGIPYAPEYKS NAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDALINSTKIYSYFPSVISKVNQGAQGILFL QWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNIVKQGYEGNFIGALETTGVVLLLEYIPEITL PVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYKLVKAKWLGTVNTQFQKRSYQMYRSLEYQVDA IKKIIDYEYKIYSGPDKEQIADEINNLKNKLEEKANKAMININIFMRESSRSFLVNQMINEAKKQLLEF DTQSKNILMQYIKANSKFIGITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVLEGGGGSGGG GSGGGGSALVAGCKNFFWKTFTSC 34. Protein sequence of the IgA-HNtet-CP-55T14 fusion ESNQPEKNGTATKPENSGNTTSENGQTEPEKKLELRNVSDIELYSQTNGTYRQHVSLDGIPENTDTYFV KVKSSAFKDVYIPVASITEEKRNGQSVYKITAKAEKLQQELENKYVDNFTFYLDKKAKEENTNFTSFSN LVKAINQNPSGTYHLAASLNANEVELGPDERSYIKDTFTGRLIGEKDGKNYAIYNLKKPLFENLSGATV EKLSLKNVAISGKNDIGSLANEATNGTKIKQVHVDGCVDGIITSKTKSDDDDKAGCKNFFWKTFTSCAL AGGGGSGGGGSGGGGSALALQCIKIKNEDLTFIAEKNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIV DYNLQSKITLPNDRTTPVTKGIPYAPEYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVD DALINSTKIYSYFPSVISKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNI VKQGYEGNFIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYKL VKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLKNKLEEKANKAM ININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKANSKFIGITELKKLESKINKVFSTPI PFSYSKNLDCWVDNEEDIDV 35. Protein sequence of the LHA-CT-SST14 fusion EFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPV SYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQ PDGSYRSEELNLVIIGPSADIIQFECLSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLL GAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSL QENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLT EIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTK LKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSD TNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHY LRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVS TTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLT VQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKN NINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGT LIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALVAGCKNFFWKTFTS C 36. Protein sequence of the LHA-CT-EGFv3 fusion EFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPV SYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQ PDGSYRSEELNLVIIGPSADIIQFECLSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLL GAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSL QENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLT EIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTK LKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSD TNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHY LRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVS TTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLT VQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKN NINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGT LIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSGGGGSALVDNSDPKCP LSHEGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELR 37. Protein sequence of the LHE-CT-IL6 fusion PKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYD PNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNG SQHILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSINEFIQDPAL TLMHELIHSLHGLYGAKGITTTCIITQQQNPLITNRKGINIEEFLTFGGNDLNIITVAQYNDIYTNLLN DYRKIASKLSKVQVSNPQLNPYKDIFQEKYGLDKDASGIYSVNINKFDDILKKLYSFTEFDLATKFQVK CRETYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIIKPITGRGLVKKIIRFCVDG IITSKTKSLIEGRNKALNLQCIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILN FNSESAPGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDT ALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVPYIGLALNI GNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKEV YSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELNQK VSIAMNNIDRFLTESSISYLMKIINEVKINKLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTL NNSIPFKLSSYTDDKILISYFNKFFKLEGGGGSGGGGSGGGGSGGGGSALVPPGEDSKDVAAPHRQPLT SSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIIT GLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWL QDMTTHLILRSFKEFLQSSLRALRQM 38. Protein sequence of the LHB-CT-IL8 fusion PVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDV CEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVN KLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQE NKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDP SIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFD KLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKA INKQAYEEISKEHLAVYKIQMCVDGIITSKTKSLIEGRNKALNLQCIDVDNEDLFFIADKNSFSDDLSK NERIEYNTQSNYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIF QYLYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANK SNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKN KIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKE KSNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENK LYLIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNSLEGGGGSGGGGSGGGGSALVAKELR CQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS 39. Protein sequence of the LHF-CP-GRAN4 fusion PVAINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTNPSDFDPPASLKNGSS AYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGKVLLQEISYAKPYLGNDHTPIDEFSPVTRTTSVNI KLSTNVESSMLLNLLVLGAGPDIFESCCYPVRKLIDPDVVYDPSNYGFGSINIVTFSPEYEYTFNDISG GHNSSTESFIADPAISLAHELIHALHGLYGARGVTYEETIEVKQAPLMIAEKPIRLEEFLTFGGQDLNI ITSAMKEKIYNNLLANYEKIATRLSEVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYK KLYSFTESDLANKFKVKARNTYFIKYEFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQSIKLNPKIIDS IPDKGLVEKIVKFAVENNNNNNNNNNLGCVDGIITSKTKSLIEGRDVKCDMEVSCPDGYTCCRLQSGAW GCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEALAGGGGSGGGGSGGGGSALVLQCIEVNNSELFFVAS ESSYNENDINTPKEIDDTTNLNNNYENNLDEVILDYNSQTIPQISNIENLNTLVQDNSYVPEYDSNGTS EIEEYDVVDFNVFFYLHAQKVPEGETNISLTSSIDTALLEESKDIFFSSEFIDTINKPVNAALFIDWIS KVIRDFTTEATQKSTVDKIADISLIVPYVGLALNIIIEAEKGNFEEAFELLGVGILLEFVPELTIPVIL VFTIKSYIDSYENKNKAIKAINNSLIEREAKWKEIYSWIVSNWLTRINTQFNKRKEQMYQALQNQVDAI KTAIEYKYNNYTSDEKNRLESEYNINNIEEELNKKVSLAMKNIERFMTESSISYLMKLINEAKVGKLKK YDNHVKSDLLNYILDHRSILGEQTNELSDLVTSTLNSSIPFELSSYTNDKILIIYFNRLYKT 40. Protein sequence of the LHD-CP-TGFa fusion TWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQS YYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVE KFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSN QSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLD VEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDK FNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNI ERNPALQKLSSESVVDLFTKVCVDGGGGSGGGGSADDDDKVVSHFNDCPDSHTQFCFHGTCRFLVQEDK PACVCHSGYVGARCEHADLLALAGGGGSGGGGSGGGGSALALQCIKVKNNRLPYVADKDSISQEIFENK IITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYY YLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKD TLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKI IKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKE NIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNII LVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFN 41. Protein sequence of the LHD-CP-TGFb fusion TWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQS YYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVE KFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSN QSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLD VEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDK FNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNI ERNPALQKLSSESVVDLFTKVCVDGGGGSGGGGSADDDDKALDTNYCFSSTEKNCCVRQLYIDFRKDLG WKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKV EQLSNMIVRSCKCSALAGGGGSGGGGSGGGGSALALQCIKVKNNRLPYVADKDSISQEIFENKIITDET NVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQK LSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKIS DVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIEN CLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQV ENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVD RLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFN 42. Protein sequence of the LHB-CT-TNFa fusion PVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDV CEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVN KLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQE NKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDP SIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFD KLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKA INKQAYEEISKEHLAVYKIQMCVDEEKLYDDDDKDRWGSSLQCIDVDNEDLFFIADKNSFSDDLSKNER IEYNTQSNYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYL YSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNT MDAIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKNKII KTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSN INIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYL IGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNSLEGGGGSGGGGSGGGGSALVRSSSRTPS DKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTH TISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAES GQVYFGIIAL 43. Protein sequence of the LHD-CT-SDF1 fusion TWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQS YYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVE KFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSN QSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLD VEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDK FNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNI ERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFEN KIITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSY YYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKK DTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREK IIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDK ENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNI ILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALKPVSLSY RCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKRFKM 44. Protein sequence of the LHC-CT-VEGF fusion PITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPKSG YYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVK TRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATND VGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPT IDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNK FVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNL SRNPALRKVNPENMLYLFTKFCVDAIDGRSLYNKTLQCRELLVKNTDLPFIGDISDVKTDIFLRKDINE ETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQ KLSDNVEDFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKI SDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTID NCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQ VENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEV DKLKAKVNNSFQNTIPFNIFSYINNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALAPMAEGGGQNHHE VVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMR IKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWS LPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR 

1. A polypeptide comprising: (i) a non-cytotoxic protease capable of cleaving a SNARE protein that is expressed in a cancer cell; (ii) a targeting moiety capable of binding to a binding site located on the surface of the cancer cell wherein the binding site is capable of undergoing endocytosis to be incorporated into an endosome within the cancer cell; and (iii) a translocation domain capable of translocating the protease from within an endosome, across the endosomal membrane and into the cytosol of the cancer cell; wherein the cancer cell is not a neuroendocrine tumour cell and the polypeptide is not clostridial neurotoxin.
 2. (canceled)
 3. The polypeptide of claim 1, wherein the cancer cell is selected from the group consisting of: a lung cancer cell, a renal cancer cell, a brain cancer cell, a breast cancer cell, a pancreatic cancer cell, a colorectal cancer cell, an adrenal cancer cell, an oesophageal cancer cell, a lymphoma cancer cell, a leukaemia cancer cell, an acute leukaemia cancer cell, a bladder cancer cell, a bone cancer cell, a bowel cancer cell, a cervical cancer cell, a chronic lymphocytic leukaemia cell, a Hodgkin's lymphoma cell, a liver cancer cell, a melanoma skin cancer cell, an oropharyngeal cancer cell, a myeloma cell, a prostate cancer cell, a soft tissue sarcoma cell, a gastric cancer cell, a testicular cancer cell, a uterine cancer cell and, a Kaposi sarcoma cancer cell.
 4. The polypeptide of claim 1, wherein the targeting moiety binds to a receptor selected from the group consisting of: an ErbB receptor a growth hormone-releasing hormone (GHRH) receptor; an insulin-like growth factor receptor (IGFR); a gastrin-releasing peptide (GRP) receptor; a bombesin peptide (BB) receptor; a growth hormone (GH) receptor; a vascular endothelial growth factor (VEGF) receptor; an acetylcholine (ACH) receptor; a somatostatin (SST) receptor; a cortistatin (CST) receptor; a chemokine (C-X-C motif) receptor; a neuropilin receptor; a gonadotropin-releasing hormone (GnRH) receptor; a VIP-glucagon-GRF-secretin superfamily receptor; a FLT receptor; a CHRN receptor; an EPH receptor; an EFN receptor; a DLK1 receptor, a DLL3 receptor, a FGF receptor; a JAG receptor; a LIF receptor; a NMB receptor; a NOTCH receptor; a PDGF receptor; a c-kit receptor; a TGF receptor; an endothelin receptor; a chemokine receptor; and an angiopoietin receptor.
 5. The polypeptide of claim 1, wherein the targeting moiety is selected from the group consisting of: an ErbB peptide, an adrenomedullin (ADM) peptide, an AM peptide, an AMF peptide, an amphiregulin peptide, an APRIL peptide, an artemin peptide, a betacellulin peptide, a bombesin peptide, a CTR peptide, an endothelin peptide, an erythropoietin peptide, a FGF peptide, a FSH peptide, a gastrin peptide, a gastrin releasing peptide (GRP), a GDNF peptide, a ghrelin peptide, a GHRH peptide, a G-CSF peptide, a growth hormone peptide, a HB-EGF peptide, a hepatocyte growth factor (HGF) peptide, an IL peptide, a keratinocyte growth factor peptide, a leptin peptide, a LIF peptide, an alpha-melanotropin peptide, a MGSA/GRO peptide, a NRG peptide, an oxytocin peptide, an osteopontin (OPN) peptide, a neuregulin-1 peptide, a VIP peptide, a PACAP peptide, a PDGF peptide, a prolactin peptide, a SCF peptide, a somatostatin (SST) peptide, a cortistatin (CST) peptide, a TNF peptide, a TGF peptide, a VEGF peptide, a vasopressin peptide, an angiopoietin peptide, a B-CLL peptide, a BCGF-12KD peptide, a BAFF peptide, a galanin peptide, a GDNF peptide, a GnRH peptide, an IGF-II peptide, a LH peptide, a neurotrophin peptide, a substance P peptide, a TGF-alpha peptide, an IGF peptide, an angiopoietin peptide, a CXC peptide, and a CCL peptide.
 6. The polypeptide of claim 1, wherein the non-cytotoxic protease comprises a clostridial neurotoxin L-chain or an IgA protease.
 7. The polypeptide of claim 1, wherein the translocation domain comprises a clostridial neurotoxin translocation domain. 8-12. (canceled)
 13. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 7, 10, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or
 44. 14. A nucleic acid encoding the polypeptide of claim
 1. 15. A nucleic acid encoding the polypeptide of claim 1, wherein the nucleic acid comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6 or
 9. 16. A method of suppressing a cancer in a patient, the method comprising administering to the patient an effective amount of the polypeptide of claim
 1. 17. The method of claim 16, wherein the cancer is selected from the group consisting of: lung cancer, renal cancer, brain cancer, breast cancer, pancreatic cancer, colorectal cancer, adrenal cancer, oesophageal cancer, lymphoma, leukaemia, acute leukaemia, bladder cancer, bone cancer, bowel cancer, cervical cancer, chronic lymphocytic leukaemia, Hodgkin's lymphoma, liver cancer, skin cancer, oropharyngeal cancer, myeloma, prostate cancer, gastric cancer, testicular cancer, uterine cancer, and Kaposi sarcoma.
 18. The method of claim 16, wherein the method comprises suppressing secretion from a cancer cell selected from the group consisting of: a lung cancer cell, a renal cancer cell, a brain cancer cell, a breast cancer cell, a pancreatic cancer cell, a colorectal cancer cell, an adrenal cancer cell, an oesophageal cancer cell, a lymphoma cancer cell, a leukaemia cancer cell, an acute leukaemia cancer cell, a bladder cancer cell, a bone cancer cell, a bowel cancer cell, a cervical cancer cell, a chronic lymphocytic leukaemia cell, a Hodgkin's lymphoma cell, a liver cancer cell, a skin cancer cell, an oropharyngeal cancer cell, a myeloma cell, a prostate cancer cell, a soft tissue sarcoma cell, a gastric cancer cell, a testicular cancer cell, a uterine cancer cell, and or a Kaposi sarcoma cell.
 19. The polypeptide of claim 4, wherein the targeting moiety is an antibody or antibody fragment.
 20. A method of suppressing a cancer in a patient, the method comprising administering to the patient an effective amount of the nucleic acid of claim
 14. 21. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 7, 10, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or
 44. 22. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 7, 10, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or
 44. 23. A nucleic acid encoding the polypeptide of claim 1, wherein the nucleic acid comprises a nucleic acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 6 or
 9. 24. A nucleic acid encoding the polypeptide of claim 1, wherein the said nucleic acid comprises a nucleic acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 6 or
 9. 